LISTERIA-MONOCYTOGENES DETECTION METHOD

TECHNICAL FIELD

The present invention relates to a method of specifically detecting Listeria monocytogenes and primers therefor.

BACKGROUND ART

Listeriosis is an infection caused by Listeria monocytogenes (which may be hereinafter referred to as “monocytogenes bacterium”). Among the about 10 bacterial species known for the genus Listeria, only the monocytogenes bacterium causes listeriosis in human.

In Western countries, this bacterium is regarded as a serious food-poisoning bacterium. Also in Japan, the monocytogenes bacterium is often detected from a variety of foods including meat products and dairy products. Since the monocytogenes bacterium can be killed by ordinary cooking with heat, food poisoning hardly occurs by foods requiring cooking with heat. However, since the monocytogenes bacterium grows even under low-temperature conditions, for example, in a refrigerator, the risk of food poisoning by the monocytogenes bacterium still exists even when appropriate storage is carried out at low temperature for foods eaten without cooking with heat, such as dairy products including cheese; and ham, salami, and smoked salmon.

In the official qualitative test for the monocytogenes bacterium, judgment for the monocytogenes bacterium is carried out based on formation of a colony accompanied by a milky-white halo on a selective isolation medium such as ALOA agar medium or CHROMagar medium (Non-patent Document 1). However, the genus Listeria includes halo-forming species other than the monocytogenes bacterium. Therefore, in cases of contamination with such bacteria belonging to the genus Listeria, they are judged as positive for the monocytogenes bacterium. Further, the official test using a selective isolation medium takes days to carry out the judgment since it requires several days of confirmation culture, and the confirmation culture requires skill, which is problematic.

A variety of primers for detection of the monocytogenes bacterium by real-time PCR or the like have been reported (for example, Patent Documents 1 and 2), and there are also commercially available kits. In these prior art techniques, genes associated with pathogenicity of the monocytogenes bacterium are targeted. However, since the known methods including the commercially available kits have failed to sufficiently suppress production of false negatives and false positives, they are not sufficiently satisfactory as test methods for specifically detecting only the monocytogenes bacterium.

PRIOR ART DOCUMENT(S)
Patent Document(s)

Patent Document 1: JP 2010-263873 A

Patent Document 2: JP 2007-61061 A

Non-Patent Document(S)

Non-Patent Document 1: Notification No. 1128, Article 2 of the Department of Food Safety, “Examination of Listeria monocytogenes”, Nov. 28, 2014

SUMMARY OF THE INVENTION
Problems to be Solved by the Invention

An object of the present invention is to provide means that enables detection of the monocytogenes bacterium alone distinctly from other bacteria belonging to the genus Listeria with sufficiently high accuracy.

Means for Solving the Problems

The present inventors intensively analyzed the genome of the monocytogenes bacterium to identify two genes as target regions with which the monocytogenes bacterium can be specifically detected distinctly from other bacteria belonging to the genus Listeria utilizing a nucleic acid amplification method. The present inventors studied the base sequences of these two genes in more detail, and carried out an intensive study by designing a large number of primers and using a variety of combinations of the primers for genomic DNAs of monocytogenes bacterial strains and other bacterial strains belonging to the genus Listeria. As a result, the present inventors succeeded in identification of primer setting regions for specific detection of the monocytogenes bacterium alone with high accuracy, and also in establishment of preferred particular examples of PCR primer sets and LAMP primer sets, thereby completing the present invention.

More specifically, the present invention provides a primer set for detection of Listeria monocytogenes, comprising any of the following primer sets for amplification of a partial region of the lmo0084 gene or the lmo2736 gene of Listeria monocytogenes:

(A-1) a set of a forward primer containing in its 3′-side the base sequence of SEQ ID NO:26 or a sequence which is the same as the base sequence except that not more than 4 bases are substituted at a genetic polymorphism site(s) in the base sequence, and a reverse primer containing in its 3′-side the base sequence of SEQ ID NO:30 or a sequence which is the same as the base sequence except that not more than 4 bases are substituted at a genetic polymorphism site(s) in the base sequence;

(A-2) a set of a forward primer containing in its 3′-side the base sequence of SEQ ID NO:26 or a sequence which is the same as the base sequence except that not more than 4 bases are substituted at a genetic polymorphism site(s) in the base sequence, and a reverse primer containing in its 3′-side the base sequence of SEQ ID NO:31 or a sequence which is the same as the base sequence except that not more than 4 bases are substituted at a genetic polymorphism site(s) in the base sequence;

(A-3) a set of a forward primer containing in its 3′-side the base sequence of SEQ ID NO:27 or a sequence which is the same as the base sequence except that not more than 4 bases are substituted at a genetic polymorphism site(s) in the base sequence, and a reverse primer containing in its 3′-side the base sequence of SEQ ID NO:30 or a sequence which is the same as the base sequence except that not more than 4 bases are substituted at a genetic polymorphism site(s) in the base sequence;

(A-4) a set of a forward primer containing in its 3′-side the base sequence of SEQ ID NO:27 or a sequence which is the same as the base sequence except that not more than 4 bases are substituted at a genetic polymorphism site(s) in the base sequence, and a reverse primer containing in its 3′-side the base sequence of SEQ ID NO:31 or a sequence which is the same as the base sequence except that not more than 4 bases are substituted at a genetic polymorphism site(s) in the base sequence;

(A-5) a set of a forward primer containing in its 3′-side the base sequence of SEQ ID NO:28 or a sequence which is the same as the base sequence except that not more than 4 bases are substituted at a genetic polymorphism site(s) in the base sequence, and a reverse primer containing in its 3′-side the base sequence of SEQ ID NO:30 or a sequence which is the same as the base sequence except that not more than 4 bases are substituted at a genetic polymorphism site(s) in the base sequence;

(A-6) a set of a forward primer containing in its 3′-side the base sequence of SEQ ID NO:28 or a sequence which is the same as the base sequence except that not more than 4 bases are substituted at a genetic polymorphism site(s) in the base sequence, and a reverse primer containing in its 3′-side the base sequence of SEQ ID NO:31 or a sequence which is the same as the base sequence except that not more than 4 bases are substituted at a genetic polymorphism site(s) in the base sequence;

(A-7) a set of a forward primer containing in its 3′-side the base sequence of SEQ ID NO:29 or a sequence which is the same as the base sequence except that not more than 4 bases are substituted at a genetic polymorphism site(s) in the base sequence, and a reverse primer containing in its 3′-side the base sequence of SEQ ID NO:30 or a sequence which is the same as the base sequence except that not more than 4 bases are substituted at a genetic polymorphism site(s) in the base sequence;

(A-8) a set of a forward primer containing in its 3′-side the base sequence of SEQ ID NO:29 or a sequence which is the same as the base sequence except that not more than 4 bases are substituted at a genetic polymorphism site(s) in the base sequence, and a reverse primer containing in its 3′-side the base sequence of SEQ ID NO:31 or a sequence which is the same as the base sequence except that not more than 4 bases are substituted at a genetic polymorphism site(s) in the base sequence;

(B-1) a set of a forward primer containing in its 3′-side the base sequence of SEQ ID NO:58 or a sequence which is the same as the base sequence except that not more than 4 bases are substituted at a genetic polymorphism site(s) in the base sequence, and a reverse primer containing in its 3′-side the base sequence of SEQ ID NO:59 or a sequence which is the same as the base sequence except that not more than 4 bases are substituted at a genetic polymorphism site(s) in the base sequence;

(C-1) a set of a forward primer containing in its 3′-side the base sequence of SEQ ID NO:32 or a sequence which is the same as the base sequence except that not more than 4 bases are substituted at a genetic polymorphism site(s) in the base sequence, and a reverse primer containing in its 3′-side the base sequence of SEQ ID NO:37 or a sequence which is the same as the base sequence except that not more than 4 bases are substituted at a genetic polymorphism site(s) in the base sequence;

(D-1) a set of a forward primer containing in its 3′-side the base sequence of SEQ ID NO:33 or a sequence which is the same as the base sequence except that not more than 4 bases are substituted at a genetic polymorphism site(s) in the base sequence, and a reverse primer containing in its 3′-side the base sequence of SEQ ID NO:38 or a sequence which is the same as the base sequence except that not more than 4 bases are substituted at a genetic polymorphism site(s) in the base sequence;

(E-1) a set of a forward primer containing in its 3′-side the base sequence of SEQ ID NO:34 or a sequence which is the same as the base sequence except that not more than 4 bases are substituted at a genetic polymorphism site(s) in the base sequence, and a reverse primer containing in its 3′-side the base sequence of SEQ ID NO:38 or a sequence which is the same as the base sequence except that not more than 4 bases are substituted at a genetic polymorphism site(s) in the base sequence;

(F-1) a set of a forward primer containing in its 3′-side the base sequence of SEQ ID NO:34 or a sequence which is the same as the base sequence except that not more than 4 bases are substituted at a genetic polymorphism site(s) in the base sequence, and a reverse primer containing in its 3′-side the base sequence of SEQ ID NO:39 or a sequence which is the same as the base sequence except that not more than 4 bases are substituted at a genetic polymorphism site(s) in the base sequence;

(G-1) a set of a forward primer containing in its 3′-side the base sequence of SEQ ID NO:34 or a sequence which is the same as the base sequence except that not more than 4 bases are substituted at a genetic polymorphism site(s) in the base sequence, and a reverse primer containing in its 3′-side the base sequence of SEQ ID NO:40 or a sequence which is the same as the base sequence except that not more than 4 bases are substituted at a genetic polymorphism site(s) in the base sequence;

(H-1) a set of a forward primer containing in its 3′-side the base sequence of SEQ ID NO:35 or a sequence which is the same as the base sequence except that not more than 4 bases are substituted at a genetic polymorphism site(s) in the base sequence, and a reverse primer containing in its 3′-side the base sequence of SEQ ID NO:39 or a sequence which is the same as the base sequence except that not more than 4 bases are substituted at a genetic polymorphism site(s) in the base sequence;

(I-1) a set of a forward primer containing in its 3′-side the base sequence of SEQ ID NO:35 or a sequence which is the same as the base sequence except that not more than 4 bases are substituted at a genetic polymorphism site(s) in the base sequence, and a reverse primer containing in its 3′-side the base sequence of SEQ ID NO:40 or a sequence which is the same as the base sequence except that not more than 4 bases are substituted at a genetic polymorphism site(s) in the base sequence;

(I-2) a set of a forward primer containing in its 3′-side the base sequence of SEQ ID NO:60 or a sequence which is the same as the base sequence except that not more than 4 bases are substituted at a genetic polymorphism site(s) in the base sequence, and a reverse primer containing in its 3′-side the base sequence of SEQ ID NO:61 or a sequence which is the same as the base sequence except that not more than 4 bases are substituted at a genetic polymorphism site(s) in the base sequence;

(I-3) a set of a forward primer containing in its 3′-side the base sequence of SEQ ID NO:62 or a sequence which is the same as the base sequence except that not more than 4 bases are substituted at a genetic polymorphism site(s) in the base sequence, and a reverse primer containing in its 3′-side the base sequence of SEQ ID NO:61 or a sequence which is the same as the base sequence except that not more than 4 bases are substituted at a genetic polymorphism site(s) in the base sequence;

(J-1) a set of a forward primer containing in its 3′-side the base sequence of SEQ ID NO:35 or a sequence which is the same as the base sequence except that not more than 4 bases are substituted at a genetic polymorphism site(s) in the base sequence, and a reverse primer containing in its 3′-side the base sequence of SEQ ID NO:41 or a sequence which is the same as the base sequence except that not more than 4 bases are substituted at a genetic polymorphism site(s) in the base sequence;

(K-1) a set of a forward primer containing in its 3′-side the base sequence of SEQ ID NO:36 or a sequence which is the same as the base sequence except that not more than 4 bases are substituted at a genetic polymorphism site(s) in the base sequence, and a reverse primer containing in its 3′-side the base sequence of SEQ ID NO:39 or a sequence which is the same as the base sequence except that not more than 4 bases are substituted at a genetic polymorphism site(s) in the base sequence;

(L-1) a set of a forward primer containing in its 3′-side the base sequence of SEQ ID NO:36 or a sequence which is the same as the base sequence except that not more than 4 bases are substituted at a genetic polymorphism site(s) in the base sequence, and a reverse primer containing in its 3′-side the base sequence of SEQ ID NO:40 or a sequence which is the same as the base sequence except that not more than 4 bases are substituted at a genetic polymorphism site(s) in the base sequence; and

(M-1) a set of a forward primer containing in its 3′-side the base sequence of SEQ ID NO:63 or a sequence which is the same as the base sequence except that not more than 4 bases are substituted at a genetic polymorphism site(s) in the base sequence, and a reverse primer containing in its 3′-side the base sequence of SEQ ID NO:64 or a sequence which is the same as the base sequence except that not more than 4 bases are substituted at a genetic polymorphism site(s) in the base sequence.

The present invention also provides a primer set for detection of Listeria monocytogenes, comprising any of the following sets:

(A-9) a set of a mixed forward primer containing in its 3′-side the base sequence of SEQ ID NO:67, and a mixed reverse primer containing in its 3′-side the base sequence of SEQ ID NO:69;

(A-10) a set of a mixed forward primer containing in its 3′-side the base sequence of SEQ ID NO:68, and a mixed reverse primer containing in its 3′-side the base sequence of SEQ ID NO:69;

(D-2) a set of a mixed forward primer containing in its 3′-side the base sequence of SEQ ID NO:70, and a mixed reverse primer containing in its 3′-side the base sequence of SEQ ID NO:74;

(E-2) a set of a mixed forward primer containing in its 3′-side the base sequence of SEQ ID NO:71, and a mixed reverse primer containing in its 3′-side the base sequence of SEQ ID NO:74;

(F-2) a set of a mixed forward primer containing in its 3′-side the base sequence of SEQ ID NO:71, and a mixed reverse primer containing in its 3′-side the base sequence of SEQ ID NO:75;

(G-2) a set of a mixed forward primer containing in its 3′-side the base sequence of SEQ ID NO:71, and a mixed reverse primer containing in its 3′-side the base sequence of SEQ ID NO:76;

(H-2) a set of a mixed forward primer containing in its 3′-side the base sequence of SEQ ID NO:72, and a mixed reverse primer containing in its 3′-side the base sequence of SEQ ID NO:75;

(I-4) a set of a mixed forward primer containing in its 3′-side the base sequence of SEQ ID NO:72, and a mixed reverse primer containing in its 3′-side the base sequence of SEQ ID NO:76;

(J-2) a set of a mixed forward primer containing in its 3′-side the base sequence of SEQ ID NO:72, and a reverse primer containing in its 3′-side the base sequence of SEQ ID NO:41 or a sequence which is the same as the base sequence except that not more than 4 bases are substituted at a genetic polymorphism site(s) in the base sequence;

(K-3) a set of a mixed forward primer containing in its 3′-side the base sequence of SEQ ID NO:73, and a mixed reverse primer containing in its 3′-side the base sequence of SEQ ID NO:75;

(L-2) a set of a mixed forward primer containing in its 3′-side the base sequence of SEQ ID NO:73, and a mixed reverse primer containing in its 3′-side the base sequence of SEQ ID NO:76;

(N-1) a set of a mixed forward primer containing in its 3′-side the base sequence of SEQ ID NO:71, and a reverse primer containing in its 3′-side the base sequence of SEQ ID NO:41 or a sequence which is the same as the base sequence except that not more than 4 bases are substituted at a genetic polymorphism site(s) in the base sequence; and

(O-1) a set of a mixed forward primer containing in its 3′-side the base sequence of SEQ ID NO:73, and a reverse primer containing in its 3′-side the base sequence of SEQ ID NO:41 or a sequence which is the same as the base sequence except that not more than 4 bases are substituted at a genetic polymorphism site(s) in the base sequence.

The present invention also provides a loop-mediated isothermal amplification primer set for detection of Listeria monocytogenes, comprising any of the following sets:

(i) a set of an F3 primer composed of the base sequence of SEQ ID NO:42, a B3 primer composed of the base sequence of SEQ ID NO:43, an FIP primer composed of the base sequence of SEQ ID NO:44, and a BIP primer composed of the base sequence of SEQ ID NO:45;

(ii) a set of an F3 primer composed of the base sequence of SEQ ID NO:46, a B3 primer composed of the base sequence of SEQ ID NO:47, an FIP primer composed of the base sequence of SEQ ID NO:48, and a BIP primer composed of the base sequence of SEQ ID NO:49;

(iii) a set of an F3 primer composed of the base sequence of SEQ ID NO:50, a B3 primer composed of the base sequence of SEQ ID NO:51, an FIP primer composed of the base sequence of SEQ ID NO:52, and a BIP primer composed of the base sequence of SEQ ID NO:53; and

(iv) a set of an F3 primer composed of the base sequence of SEQ ID NO:54, a B3 primer composed of the base sequence of SEQ ID NO:55, an FIP primer composed of the base sequence of SEQ ID NO:56, and a BIP primer composed of the base sequence of SEQ ID NO:57.

The present invention also provides a method of detecting Listeria monocytogenes, comprising a step of amplifying a partial region of the lmo0084 gene or the lmo2736 gene by a nucleic acid amplification method using the primer set of the present invention described above.

The present invention also provides a probe for detection of Listeria monocytogenes, comprising an oligonucleotide portion having the base sequence of SEQ ID NO:77, SEQ ID NOs:80 to 82, SEQ ID NO:85 (wherein ngaan is tgaaa or cgaac), or SEQ ID NO:86 (wherein ngcaan is ggcaag or cgcaac).

The present invention also provides a primer-probe set for real-time PCR for detection of Listeria monocytogenes, comprising any of the following sets of primers and a probe:

[1] a set of a forward primer containing in its 3′-side the base sequence of SEQ ID NO:67, a reverse primer containing in its 3′-side the base sequence of SEQ ID NO:69, and a probe containing an oligonucleotide portion having the base sequence of SEQ ID NO:77 or 80;

[2] a set of a forward primer containing in its 3′-side the base sequence of SEQ ID NO:67, a reverse primer containing in its 3′-side the base sequence of SEQ ID NO:69, and a mixed probe containing an oligonucleotide portion having the base sequence of SEQ ID NO:85 (wherein ngaan is tgaaa or cgaac);

[3] a set of a forward primer containing in its 3′-side the base sequence of SEQ ID NO:68, a reverse primer containing in its 3′-side the base sequence of SEQ ID NO:69, and a probe containing an oligonucleotide portion having the base sequence of SEQ ID NO:77 or 80;

[4] a set of a forward primer containing in its 3′-side the base sequence of SEQ ID NO:68, a reverse primer containing in its 3′-side the base sequence of SEQ ID NO:69, and a mixed probe containing an oligonucleotide portion having the base sequence of SEQ ID NO:85 (wherein ngaan is tgaaa or cgaac);

[5] a set of a forward primer containing in its 3′-side the base sequence of SEQ ID NO:32, a reverse primer containing in its 3′-side the base sequence of SEQ ID NO:37, and a probe containing an oligonucleotide portion having the base sequence of SEQ ID NO:81; and

[6] a set of a forward primer containing in its 3′-side the base sequence of SEQ ID NO:70, a reverse primer containing in its 3′-side the base sequence of SEQ ID NO:74, and a probe containing an oligonucleotide portion having the base sequence of SEQ ID NO:82; and

[7] a set of a forward primer containing in its 3′-side the base sequence of SEQ ID NO:72, a reverse primer containing in its 3′-side the base sequence of SEQ ID NO:76, and a mixed probe containing an oligonucleotide portion having the base sequence of SEQ ID NO:86 (wherein ngcaan is ggcaag or cgcaac).

Effect of the Invention

According to the present invention, primers with which various bacterial strains of the monocytogenes bacterium can be specifically detected distinctly from other bacteria belonging to the genus Listeria are provided. According to the method of the present invention, occurrence of false negatives and false positives can be remarkably reduced compared to test methods based on conventional nucleic acid amplification methods. Bacteria belonging to the genus Listeria also include species other than the monocytogenes bacterium that form colonies accompanied by milky-white halos on a selective isolation medium. According to the present invention, no amplification occurs with those bacterial strains, and such bacterial strains can therefore be distinguished from the monocytogenes bacterium even based on the result of a nucleic acid amplification method alone. Further, serotypes of the monocytogenes bacterium can be identified by designing probes targeting polymorphic sequences characteristic to the individual serotypes, such as the TaqMan (registered trademark) probe 0084TMP535-558(CC) in the following Examples, and carrying out real-time PCR.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1-1 shows images of colonies obtained by culturing the monocytogenes bacterium on ALOA agar medium or CHROMagar medium (examples of images of a positive colony forming a halo or a false-negative colony forming no halo).

FIG. 1-2 shows images of colonies obtained by culturing species belonging to the genus Listeria other than the monocytogenes bacterium on ALOA agar medium or CHROMagar medium (examples of images of a false-positive colony forming a halo).

FIG. 1-3 shows images of colonies obtained by culturing species belonging to the genus Listeria other than the monocytogenes bacterium on ALOA agar medium or CHROMagar medium (examples of images of a negative colony forming no halo).

FIG. 2-1 shows an example of the result of PCR using PCR primers for detection of Listeria monocytogenes designed in Examples. The PCR was carried out using Prime set No. 4, which targets the lmo00084 gene. Detection was carried out by 2% agarose gel electrophoresis. The 476-bp PCR products indicated by arrows are specific amplification products from the monocytogenes bacterium. The number assigned to each lane corresponds to a bacterial strain No. listed in Table 5-1 or Table 5-2. The numbers 1 to 10 correspond to monocytogenes (corresponding, from No. 1, to the serotypes 1/2a, 1/2b, 1/2c, 3a, 3b, 3c, 4a, 4b, 4d, and 5 in this order), and the numbers 11 to 22 correspond to species belonging to the genus Listeria other than monocytogenes.

FIG. 2-2 shows an example of the result of PCR using PCR primers for detection of Listeria monocytogenes designed in Examples. The PCR was carried out using Prime set No. 1, which targets the lmo02736 gene. Detection was carried out by 2% agarose gel electrophoresis. The 168-bp PCR products indicated by arrows are specific amplification products from the monocytogenes bacterium. The arrows indicate the specific amplification products. The number assigned to each lane is the same as in FIG. 2-1.

MODE FOR CARRYING OUT THE INVENTION

One of the following two genes present in the genome of the monocytogenes bacterium is the target to be detected in the present invention.

TABLE 1

Gene name (*)
Gene length
Gene type
Description

lmo0084
984
CDS
similar to oxidoreductases

lmo2736
1134
CDS
conserved hypothetical protein

(*) In the genomic sequence information of GenBank Accession No. AL591824.1, the lmo0084 gene corresponds to the region of 86747-87744, and lmo2736 corresponds to the region of 2811788-2812921.

SEQ ID NOs:1 to 12 in SEQUENCE LISTING show base sequences of the lmo0084 gene in the serotypes 1/2a, 1/2b, 1/2c, 3a, 3b, 3c, 4a, 4b, 4c, 4d, 4e, and 7, respectively, of the monocytogenes bacterium. SEQ ID NOs:13 to 25 show base sequences of the lmo2736 gene of the above individual serotypes of the monocytogenes bacterium (regarding 4b, two kinds of base sequences are shown as SEQ ID NOs:20 and 21). In the present description, specification of partial regions of each gene is carried out using, as a standard, the base sequence in the serotype 1/2a shown in SEQ ID NO:1 for the lmo0084 gene, or the base sequence in the serotype 1/2a shown in SEQ ID NO:13 for the lmo2736 gene. For example, “the region of position 306 to position 737 in the lmo0084 gene shown in SEQ ID NO:1” includes the region of position 306 to position 737 in the lmo0084 gene of various serotypes. The same applies to the lmo2736 gene. The accession numbers of the sequences of SEQ ID NOs:1 to 25 are as shown below in Table 2.

TABLE 2

SEQ ID NO.
Gene name
Serotype
Accession No.

1
lmo0084
1/2a
NC_018592.1

2
lmo0084
1/2b
NC_018587.1

3
lmo0084
1/2c
NC_018588.1

4
lmo0084
3a
NC_018593.1

5
lmo0084
3b
NC_018586.1

6
lmo0084
3c
NC_018589.1

7
lmo0084
4a
NC_017529.1

8
lmo0084
4b
NC_019556.1

9
lmo0084
4c
NC_018590.1

10
lmo0084
4d
NC_018584.1

11
lmo0084
4e
NC_018585.1

12
lmo0084
7
NC_018591.1

13
lmo2736
1/2a
NC_018592.1

14
lmo2736
1/2b
NC_018587.1

15
lmo2736
1/2c
NC_018588.1

16
lmo2736
3a
NC_018593.1

17
lmo2736
3b
NC_018586.1

18
lmo2736
3c
NC_018589.1

19
lmo2736
4a
NC_017529.1

20
lmo2736
4b
NC_019556.1

21
lmo2736
4b
NC_018642.1

22
lmo2736
4c
NC_018590.1

23
lmo2736
4d
NC_018584.1

24
lmo2736
4e
NC_018585.1

25
lmo2736
7
NC_018591.1

The specific detection of the monocytogenes bacterium can be carried out by a nucleic acid amplification method using a primer(s) for detection of Listeria monocytogenes, which primer(s) specifically hybridize(s) to a region in the lmo0084 gene or the lmo2736 gene. As the nucleic acid amplification method, various known methods such as the PCR method or the isothermal amplification method may be used. In the present invention, the term “primer” includes PCR primers and isothermal amplification primers. In the present invention, the PCR method means a nucleic acid amplification method in which the temperature is repeatedly changed to amplify a region of interest.

The term “specifically hybridizes” means that, under normal hybridization conditions, the primer hybridizes only to a target region, and does not substantially hybridize to other regions. The term “under normal hybridization conditions” means that a reaction is carried out under conditions used for annealing in normal PCR, for example, at an appropriate annealing temperature of about 54° C. to 60° C. using a common buffer such as 50 mM KCl, 10 mM Tris-HCl (pH 8.3 to 9.0), 1.5 mM MgCl₂in cases of PCR using Taq polymerase. However, the appropriate annealing temperature is not limited to the above example, and may be determined based on the Tm value of the primer and an empirical rule by the experimenter. Those skilled in the art can easily determine the temperature. The term “does not substantially hybridize” means that the primer does not hybridize at all, or, even in cases where it hybridizes, a much smaller amount of the primer hybridizes compared to the case where the primer hybridizes to the target region, so that only a relatively ignorable, small amount of the primer hybridizes.

For detection of the amplification product obtained by the nucleic acid amplification method, any known detection method may be applied. In cases of the PCR method, the detection may be carried out by electrophoresis, the intercalation method, the quencher-mediated fluorescence detection method, or the like, and, in cases of the isothermal amplification method, the detection may be carried out by a method in which pyrophosphoric acid as an amplification by-product is insolubilized, the intercalation method, the quencher-mediated fluorescence detection method, or the like. Alternatively, the amplification product may be detected by nucleic acid chromatography.

The term “PCR method” also includes the real-time PCR method. In real-time PCR, detection and monitoring of the amplification product are commonly carried out by the intercalation method or the quencher-mediated fluorescence detection method. In the following Examples, a specific example of the real-time PCR detection system using the TaqMan (registered trademark) probe method as one example of the quencher-mediated fluorescence detection method is described. However, the detection method is not limited thereto, and a variety of methods may be employed.

In cases of nucleic acid chromatography, the detection is possible by carrying out nucleic acid amplification using a primer set for detection of the monocytogenes bacterium of the invention, and then developing the resulting amplification product on a strip on which a capture substance that specifically binds to the amplification product is immobilized in the shape of a line or the like. For capturing the amplification product, for example, a labeling compound such as biotin or DIG, or an arbitrary base sequence may be added to the 5-side of the forward or reverse primer, and a labeling-compound-specific binding substance such as avidin or an anti-DIG antibody, or an oligonucleotide probe having a base sequence complementary to the arbitrary base sequence may be immobilized as the capture substance on the strip. For further increasing the specificity of the detection, as the capture probe on the strip, a probe having a base sequence that specifically hybridizes to a certain partial sequence in the region amplified by the primers may be used. In order to provide such a capture probe, a partial region in the region amplified by the primers may be appropriately selected, and a probe capable of hybridizing to the amplification product of each serotype may be designed with reference to the base sequence of the lmo0084 gene of each serotype of SEQ ID NOs:1 to 12 or the base sequence of the lmo2736 gene of each serotype of SEQ ID NOs:13 to 25. The detection system may be constructed in the same manner as in a known nucleic acid chromatography method, and examples of the detection system include coloring detection methods using an enzyme such as peroxidase or using particles such as colloidal gold or colored latex.

The isothermal amplification method is not limited, and various isothermal amplification methods such as the Loop-Mediated Isothermal Amplification (LAMP) method, the Strand Displacement Amplification (SDA) method, the Isothermal and Chimeric primer-initiated Amplification of Nucleic acids (ICAN) method, the Helicase-Dependent Amplification (HDA) method, and the Nicking Enzyme Amplification Reaction (NEAR) method may be employed. Examples of the isothermal amplification primers include the LAMP primers designed in the following Examples.

In the present invention, typical samples to be tested are samples collected from foods (including raw materials and processed foods). However, the samples to be tested are not limited thereto, and include a variety of samples whose test for the monocytogenes bacterium is desired, such as swabs from production lines and fingers of workers in food factories.

As a primer set to be used for the nucleic acid amplification method such as the PCR method or the isothermal amplification method for specifically detecting the monocytogenes bacterium distinctly from other bacteria belonging to the genus Listeria and from other food-poisoning microbes, a primer set which specifically hybridizes to a region in the lmo0084 gene sequence of SEQ ID NO:1 or the lmo2736 gene sequence of SEQ ID NO:13 may be used. The primer set may be designed taking into account the primer length, the GC content, the Tm value, bias of bases, contiguous sequences, complementarity inside and between primers, the molecular weight of the amplification product, genetic polymorphisms in the target region, and the like. In cases where the primer set is used in the PCR method, it may be designed to have a length of about 15 to 30 bases, a GC content of about 40 to 60%, and a Tm value of about 50 to 70° C. For a nucleic acid amplification method other than the PCR method, the primer set may be designed according to the principle of the method, for example, as in the LAMP method described below.

Each primer constituting such a primer set is generally preferably designed for a region having less sequence diversity among serotypes, but may also be designed in a region having a small number of genetic polymorphisms. In cases where the primer is designed for a region containing a genetic polymorphism(s), the primer may be designed such that a base substitution(s) reflecting the genetic polymorphism(s) is/are added to the gene sequence in the serotype 1/2a of SEQ ID NO:1 or SEQ ID NO:13. The number of the base substitution(s) reflecting the genetic polymorphism(s) is preferably not more than 20%, more preferably not more than 15% per primer. More specifically, in cases of a primer having a chain length of 20 bases containing no additional sequence, the primer may be designed to have a sequence in which not more than 4, preferably not more than 3 bases are substituted at a genetic polymorphism site(s) in the 20-base region. In some cases, the thus designed primer may have a sequence identical to the sequence of a partial region of the gene sequence of another serotype, or the complementary strand thereof. In cases where primers containing substitutions at a genetic polymorphism site(s) are used, primers for the individual genetic polymorphisms may be used; a primer mixture prepared by mixing the primers for the individual genetic polymorphisms may be used; or a mixed primer synthesized such that the genetic polymorphism site(s) has/have mixed bases according to the genetic polymorphisms (for example, when some serotypes have G while other serotypes have C as the base at a certain site, a mixed primer prepared such that the base at the site is S (G or C)); may be used.

In the present invention, a primer for specifically detecting the monocytogenes bacterium may be designed such that the primer specifically hybridizes to any of the following regions (1) to (14) taking the above factors into account.

(1) The region of position 261 to position 325 of the lmo0084 gene sequence of SEQ ID NO:1, or the region complementary to this region.

LMO0084-F286A (SEQ ID NO:26), LMO0084-F286B (SEQ ID NO:27), LMO0084-F281A (SEQ ID NO:28), and LMO0084-F281B (SEQ ID NO:29) in Examples are specific examples of a forward primer that hybridizes to the region complementary to the region of position 261 to position 325. LMO0084-F286/M (SEQ ID NO:67) and LMO0084-F281/M (SEQ ID NO:68) are specific examples of a mixed forward primer that hybridizes to the region complementary to this region. Primers containing the base sequence of SEQ ID NO:59 in the 3″-side thereof, such as the LAMP primer LMO84 BIP (SEQ ID NO:45) in Examples, are specific examples of a reverse primer that hybridizes to the region of position 261 to position 325.

(2) The region of position 718 to position 777 of the lmo0084 gene sequence of SEQ ID NO:1, or the region complementary to this region.

LMO0084-R757A (SEQ ID NO:30) and LMO0084-R757B (SEQ ID NO:31) in Examples are specific examples of a reverse primer that hybridizes to the region of position 718 to position 777. LMO0084-R757/M (SEQ ID NO:69) is a specific example of a mixed reverse primer that hybridizes to this region.

(3) The region of position 108 to position 166 of the lmo0084 gene sequence of SEQ ID NO:1, or the region complementary to this region.

Primers containing the base sequence of SEQ ID NO:58 in the 3′-side thereof, such as the LAMP primer LMO84 FIP (SEQ ID NO:44) in Examples, are specific examples of a forward primer that hybridizes to the region complementary to the region of position 108 to position 166.

(4) The region of position 1 to position 47 of the lmo2736 gene sequence of SEQ ID NO:13, or the region complementary to this region.

LMO2736-F8 (SEQ ID NO:32) in Examples is a specific example of a forward primer that hybridizes to the region complementary to the region of position 1 to position 47.

(5) The region of position 202 to position 261 of the lmo2736 gene sequence of SEQ ID NO:13, or the region complementary to this region.

LMO2736-F222 (SEQ ID NO:33) in Examples is a specific example of a forward primer that hybridizes to the region complementary to the region of position 202 to position 261. LMO2736-F222/M (SEQ ID NO:70) is a specific example of a mixed forward primer that hybridizes to the region complementary to this region.

(6) The region of position 468 to position 527 of the lmo2736 gene sequence of SEQ ID NO:13, or the region complementary to this region.

LMO2736-F488 (SEQ ID NO:34) in Examples is a specific example of a forward primer that hybridizes to the region complementary to the region of position 468 to position 527. LMO2736-F488/M (SEQ ID NO:71) is a specific example of a mixed forward primer that hybridizes to the region complementary to this region.

(7) The region of position 510 to position 569 of the lmo2736 gene sequence of SEQ ID NO:13, or the region complementary to this region.

LMO2736-F530 (SEQ ID NO:35) in Examples, and primers containing the base sequence of SEQ ID NO:60 or 62 in the 3′-side thereof, such as LMO2736-1 FIP (SEQ ID NO:48) and LMO2736-2 FIP (SEQ ID NO:52) in Examples, are specific examples of a forward primer that hybridizes to the region complementary to the region of position 510 to position 569. LMO2736-F530/M (SEQ ID NO:72) is a specific example of a mixed forward primer that hybridizes to the region complementary to this region.

(8) The region of position 552 to position 611 of the lmo2736 gene sequence of SEQ ID NO:13, or the region complementary to this region.

LMO2736-F572 (SEQ ID NO:36) is a specific example of a forward primer that hybridizes to the region complementary to the region of position 552 to position 611; LMO2736-F572/M (SEQ ID NO:73) is a specific example of a mixed forward primer that hybridizes to the region complementary to this region; and LMO2736-R591 (SEQ ID NO:38) is a specific example of a reverse primer that hybridizes to the region of position 552 to position 611.

(9) The region of position 137 to position 196 of the lmo2736 gene sequence of SEQ ID NO:13, or the region complementary to this region.

LMO2736-R176 (SEQ ID NO:37) in Examples is a specific example of a reverse primer that hybridizes to the region of position 137 to position 196.

(10) The region of position 646 to position 705 of the lmo2736 gene sequence of SEQ ID NO:13, or the region complementary to this region.

LMO2736-R685 (SEQ ID NO:39) in Examples is a specific example of a reverse primer that hybridizes to the region of position 646 to position 705. LMO2736-R685/M (SEQ ID NO:75) is a specific example of a mixed reverse primer that hybridizes to this region.

(11) The region of position 732 to position 791 of the lmo2736 gene sequence of SEQ ID NO:13, or the region complementary to this region.

LMO2736-R771 (SEQ ID NO:40) in Examples, and primers containing the base sequence of SEQ ID NO:61 in the 3′-side thereof, such as LMO2736-1 BIP (SEQ ID NO:49) and LMO2736-2 BIP (SEQ ID NO:53) in Examples, are specific examples of a reverse primer that hybridizes to the region of position 732 to position 791. LMO2736-R771/M (SEQ ID NO:76) is a specific example of a mixed reverse primer that hybridizes to this region.

(12) The region of position 953 to position 1012 of the lmo2736 gene sequence of SEQ ID NO:13, or the region complementary to this region.

LMO2736-R992 (SEQ ID NO:41) in Examples is a specific example of a reverse primer that hybridizes to the region of position 953 to position 1012.

(13) The region of position 496 to position 560 of the lmo2736 gene sequence of SEQ ID NO:13, or the region complementary to this region.

Primers containing the base sequence of SEQ ID NO:63 in the 3′-side thereof, such as the LAMP primer LMO2736-10 FIP (SEQ ID NO:56) in Examples, are specific examples of a forward primer that hybridizes to the region complementary to the region of position 496 to position 560.

(14) The region of position 721 to position 775 of the lmo2736 gene sequence of SEQ ID NO:13, or the region complementary to this region.

Primers containing the base sequence of SEQ ID NO:64 in the 3′-side thereof, such as the LAMP primer LMO2736-10 BIP (SEQ ID NO:57) in Examples, are specific examples of a reverse primer that hybridizes to the region of position 721 to position 775.

The primers that specifically hybridize to the regions (1) to (3) of the lmo0084 gene may be, for example, primers each containing in its 3′-side a sequence having not less than 15 consecutive bases, preferably not less than 18 consecutive bases in the region of position 261 to position 325, the region of position 718 to position 777, or the region of position 108 to position 166 in the base sequence of SEQ ID NO:1, or in the region complementary to any of these; or a sequence which is the same as this sequence except that not more than 20% of bases are substituted at a genetic polymorphism site(s) therein.

The primers that specifically hybridize to the regions (4) to (14) of the lmo2736 gene may be, for example, primers each containing in its 3′-side a sequence having not less than 15 consecutive bases, preferably not less than 18 consecutive bases in the region of position 1 to position 47, the region of position 202 to position 261, the region of position 468 to position 527, the region of position 510 to position 569, the region of position 552 to position 611, the region of position 137 to position 196, the region of position 646 to position 705, the region of position 732 to position 791, the region of position 953 to position 1012, the region of position 496 to position 560, or the region of position 721 to position 775 in the base sequence of SEQ ID NO:13, or in the region complementary to any of these; or a sequence which is the same as this sequence except that not more than 20% of bases are substituted at a genetic polymorphism site(s) therein.

Specific examples of a preferred sequence that can be employed for a primer for amplifying/detecting a partial region of the lmo0084 gene include SEQ ID NOs:26 to 31, 58, and 59. SEQ ID NOs:58 and 59 are 3′-side partial sequences of SEQ ID NOs:44 and 45, which are LAMP primer sequences (sequences of the F2 or B2 portion, which hybridize to target sites in the lmo0084 gene). SEQ ID NOs:26 to 29 and 58 are sequences of the sense strand of the lmo0084 gene, and can be used as the sequences of forward primers that hybridize to the antisense strand of the gene. SEQ ID NOs:30, 31, and 59 are sequences of the antisense strand of the lmo0084 gene, and can be used as sequences of reverse primers that hybridize to the sense strand of the gene.

Specific examples of a preferred sequence that can be employed for a primer for amplifying/detecting a partial region of the 1=2736 gene include SEQ ID NOs:32 to 41, and 60 to 64. SEQ ID NOs:60 to 64 are 3′-side partial sequences of SEQ ID NOs:48, 49, 52, 53, 56, and 57, which are LAMP primer sequences (sequences of the F2 or B2 portion, which hybridize to target sites in the lmo2736 gene). SEQ ID NOs:32 to 36, 60, 62, and 63 are sequences of the sense strand of the lmo2736 gene, and can be used as the sequences of forward primers that hybridize to the antisense strand of the gene. SEQ ID NOs:37 to 41, 61, and 64 are sequences of the antisense strand of the lmo2736 gene, and can be used as sequences of reverse primers that hybridize to the sense strand of the gene.

SEQ ID NOs:26 to 31 and SEQ ID NOs:32 to 41, which were mentioned as preferred specific examples of sequences that can be employed for primers for amplifying/detecting a partial region of the lmo0084 gene or the lmo2736 gene, can be used as LAMP primers by providing an additional sequence to the 5′-side thereof as described below.

Examples of the set of a forward primer and a reverse primer for amplification of a partial region of the lmo0084 gene or the lmo2736 gene of the monocytogenes bacterium, designed for the regions (1) to (14), include primer sets containing any of the following. The primer set may be PCR primers, or isothermal amplification primers such as LAMP primers.

(A) A set of a forward primer that hybridizes to the region (1) and a reverse primer that hybridizes to the region (2).

(B) A set of a forward primer that hybridizes to the region (3) and a reverse primer that hybridizes to the region (1).

(C) A set of a forward primer that hybridizes to the region (4) and a reverse primer that hybridizes to the region (9).

(D) A set of a forward primer that hybridizes to the region (5) and a reverse primer that hybridizes to the region (8).

(E) A set of a forward primer that hybridizes to the region (6) and a reverse primer that hybridizes to the region (8).

(F) A set of a forward primer that hybridizes to the region (6) and a reverse primer that hybridizes to the region (10).

(G) A set of a forward primer that hybridizes to the region (6) and a reverse primer that hybridizes to the region (11).

(H) A set of a forward primer that hybridizes to the region (7) and a reverse primer that hybridizes to the region (10).

(I) A set of a forward primer that hybridizes to the region (7) and a reverse primer that hybridizes to the region (11).

(J) A set of a forward primer that hybridizes to the region (7) and a reverse primer that hybridizes to the region (12).

(K) A set of a forward primer that hybridizes to the region (8) and a reverse primer that hybridizes to the region (10).

(L) A set of a forward primer that hybridizes to the region (8) and a reverse primer that hybridizes to the region (11).

(M) A set of a forward primer that hybridizes to the region (13) and a reverse primer that hybridizes to the region (14).

(N) A set of a forward primer that hybridizes to the region (6) and a reverse primer that hybridizes to the region (12).

(O) A set of a forward primer that hybridizes to the region (8) and a reverse primer that hybridizes to the region (12).

Specific examples of the sets (A) to (O) described above include the following sets. The alphabets correspond to the (A) to (O), respectively. For example, the following (A-1) to (A-10) are examples of the set (A).

(A-1) A set of a forward primer containing in its 3′-side a sequence having not less than 15 consecutive bases, preferably not less than 18 consecutive bases in the base sequence of SEQ ID NO:26, more preferably the full-length sequence of the base sequence, or a sequence which is the same as the base sequence of SEQ ID NO:26 except that not more than 4 bases are substituted at a genetic polymorphism site(s) therein, and a reverse primer containing in its 3′-side a sequence having not less than 15 consecutive bases, preferably not less than 18 consecutive bases in the base sequence of SEQ ID NO:30, more preferably the full-length sequence of the base sequence, or a sequence which is the same as the base sequence of SEQ ID NO:30 except that not more than 4 bases are substituted at a genetic polymorphism site(s) therein. Specific examples of this set include the set of LMO0084-F286A and LMO0084-R757A in the Examples described below.

(A-2) A set of a forward primer containing in its 3′-side a sequence having not less than 15 consecutive bases, preferably not less than 18 consecutive bases in the base sequence of SEQ ID NO:26, more preferably the full-length sequence of the base sequence, or a sequence which is the same as the base sequence of SEQ ID NO:26 except that not more than 4 bases are substituted at a genetic polymorphism site(s) therein, and a reverse primer containing in its 3′-side a sequence having not less than 15 consecutive bases, preferably not less than 18 consecutive bases in the base sequence of SEQ ID NO:31, more preferably the full-length sequence of the base sequence, or a sequence which is the same as the base sequence of SEQ ID NO:31 except that not more than 4 bases are substituted at a genetic polymorphism site(s) therein. Specific examples of this set include the set of LMO0084-F286A and LMO0084-R757B in the Examples described below.

(A-3) A set of a forward primer containing in its 3′-side a sequence having not less than 15 consecutive bases, preferably not less than 18 consecutive bases in the base sequence of SEQ ID NO:27, more preferably the full-length sequence of the base sequence, or a sequence which is the same as the base sequence of SEQ ID NO:27 except that not more than 4 bases are substituted at a genetic polymorphism site(s) therein, and a reverse primer containing in its 3′-side a sequence having not less than 15 consecutive bases, preferably not less than 18 consecutive bases in the base sequence of SEQ ID NO:30, more preferably the full-length sequence of the base sequence, or a sequence which is the same as the base sequence of SEQ ID NO:30 except that not more than 4 bases are substituted at a genetic polymorphism site(s) therein. Specific examples of this set include the set of LMO0084-F286B and LMO0084-R757A in the Examples described below.

(A-4) A set of a forward primer containing in its 3′-side a sequence having not less than 15 consecutive bases, preferably not less than 18 consecutive bases in the base sequence of SEQ ID NO:27, more preferably the full-length sequence of the base sequence, or a sequence which is the same as the base sequence of SEQ ID NO:27 except that not more than 4 bases are substituted at a genetic polymorphism site(s) therein, and a reverse primer containing in its 3′-side a sequence having not less than 15 consecutive bases, preferably not less than 18 consecutive bases in the base sequence of SEQ ID NO:31, more preferably the full-length sequence of the base sequence, or a sequence which is the same as the base sequence of SEQ ID NO:31 except that not more than 4 bases are substituted at a genetic polymorphism site(s) therein. Specific examples of this set include the set of LMO0084-F286B and LMO0084-R757B in the Examples described below.

(A-5) A set of a forward primer containing in its 3′-side a sequence having not less than 15 consecutive bases, preferably not less than 18 consecutive bases in the base sequence of SEQ ID NO:28, more preferably the full-length sequence of the base sequence, or a sequence which is the same as the base sequence of SEQ ID NO:28 except that not more than 4 bases are substituted at a genetic polymorphism site(s) therein, and a reverse primer containing in its 3′-side a sequence having not less than 15 consecutive bases, preferably not less than 18 consecutive bases in the base sequence of SEQ ID NO:30, more preferably the full-length sequence of the base sequence, or a sequence which is the same as the base sequence of SEQ ID NO:30 except that not more than 4 bases are substituted at a genetic polymorphism site(s) therein. Specific examples of this set include the set of LMO0084-F281A and LMO0084-R757A in the Examples described below.

(A-6) A set of a forward primer containing in its 3′-side a sequence having not less than 15 consecutive bases, preferably not less than 18 consecutive bases in the base sequence of SEQ ID NO:28, more preferably the full-length sequence of the base sequence, or a sequence which is the same as the base sequence of SEQ ID NO:28 except that not more than 4 bases are substituted at a genetic polymorphism site(s) therein, and a reverse primer containing in its 3′-side a sequence having not less than consecutive bases, preferably not less than 18 consecutive bases in the base sequence of SEQ ID NO:31, more preferably the full-length sequence of the base sequence, or a sequence which is the same as the base sequence of SEQ ID NO:31 except that not more than 4 bases are substituted at a genetic polymorphism site(s) therein. Specific examples of this set include the set of LMO0084-F281A and LMO0084-R757B in the Examples described below.

(A-7) A set of a forward primer containing in its 3′-side a sequence having not less than 15 consecutive bases, preferably not less than 18 consecutive bases in the base sequence of SEQ ID NO:29, more preferably the full-length sequence of the base sequence, or a sequence which is the same as the base sequence of SEQ ID NO:29 except that not more than 4 bases are substituted at a genetic polymorphism site(s) therein, and a reverse primer containing in its 3′-side a sequence having not less than 15 consecutive bases, preferably not less than 18 consecutive bases in the base sequence of SEQ ID NO:30, more preferably the full-length sequence of the base sequence, or a sequence which is the same as the base sequence of SEQ ID NO:30 except that not more than 4 bases are substituted at a genetic polymorphism site(s) therein. Specific examples of this set include the set of LMO0084-F281B and LMO0084-R757A in the Examples described below.

(A-8) A set of a forward primer containing in its 3′-side a sequence having not less than 15 consecutive bases, preferably not less than 18 consecutive bases in the base sequence of SEQ ID NO:29, more preferably the full-length sequence of the base sequence, or a sequence which is the same as the base sequence of SEQ ID NO:29 except that not more than 4 bases are substituted at a genetic polymorphism site(s) therein, and a reverse primer containing in its 3′-side a sequence having not less than 15 consecutive bases, preferably not less than 18 consecutive bases in the base sequence of SEQ ID NO:31, more preferably the full-length sequence of the base sequence, or a sequence which is the same as the base sequence of SEQ ID NO:31 except that not more than 4 bases are substituted at a genetic polymorphism site(s) therein. Specific examples of this set include the set of LMO0084-F281B and LMO0084-R757B in the Examples described below.

(A-9) A set of a mixed forward primer containing in its 3′-side a sequence having not less than 15 consecutive bases, preferably not less than 18 consecutive bases in the base sequence of SEQ ID NO:67, more preferably the full-length sequence of the base sequence, and a mixed reverse primer containing in its 3′-side a sequence having not less than 15 consecutive bases, preferably not less than 18 consecutive bases in the base sequence of SEQ ID NO:69, more preferably the full-length sequence of the base sequence. Specific examples of this set include the set of LMO0084-F286/M and LMO0084-R757/M in the Examples described below.

(A-10) A set of a mixed forward primer containing in its 3′-side a sequence having not less than 15 consecutive bases, preferably not less than 18 consecutive bases in the base sequence of SEQ ID NO:68, more preferably the full-length sequence of the base sequence, and a mixed reverse primer containing in its 3′-side a sequence having not less than 15 consecutive bases, preferably not less than 18 consecutive bases in the base sequence of SEQ ID NO:69, more preferably the full-length sequence of the base sequence. Specific examples of this set include the set of LMO0084-F281/M and LMO0084-R757/M in the Examples described below.

(B-1) A set of a forward primer containing in its 3′-side a sequence having not less than 15 consecutive bases, preferably not less than 18 consecutive bases in the base sequence of SEQ ID NO:58, more preferably the full-length sequence of the base sequence, or a sequence which is the same as the base sequence of SEQ ID NO:58 except that not more than 4 bases are substituted at a genetic polymorphism site(s) therein, and a reverse primer containing in its 3′-side a sequence having not less than 15 consecutive bases in the base sequence of SEQ ID NO:59, preferably the full-length sequence of the base sequence, or a sequence which is the same as the base sequence of SEQ ID NO:59 except that not more than 4 bases are substituted at a genetic polymorphism site(s) therein. Specific examples of this set include the set of the LAMP primers LMO84 FIP and LMO84 BIP in the Examples described below.

(C-1) A set of a forward primer containing in its 3′-side a sequence having not less than 15 consecutive bases, preferably not less than 18 consecutive bases in the base sequence of SEQ ID NO:32, more preferably the full-length sequence of the base sequence, or a sequence which is the same as the base sequence of SEQ ID NO:32 except that not more than 4 bases are substituted at a genetic polymorphism site(s) therein, and a reverse primer containing in its 3′-side a sequence having not less than 15 consecutive bases, preferably not less than 18 consecutive bases in the base sequence of SEQ ID NO:37, more preferably the full-length sequence of the base sequence, or a sequence which is the same as the base sequence of SEQ ID NO:37 except that not more than 4 bases are substituted at a genetic polymorphism site(s) therein. Specific examples of this set include the set of LMO2736-F8 and LMO2736-R176 in the Examples described below.

(D-1) A set of a forward primer containing in its 3′-side a sequence having not less than 15 consecutive bases, preferably not less than 18 consecutive bases in the base sequence of SEQ ID NO:33, more preferably the full-length sequence of the base sequence, or a sequence which is the same as the base sequence of SEQ ID NO:33 except that not more than 4 bases are substituted at a genetic polymorphism site(s) therein, and a reverse primer containing in its 3′-side a sequence having not less than 15 consecutive bases, preferably not less than 18 consecutive bases in the base sequence of SEQ ID NO:38, more preferably the full-length sequence of the base sequence, or a sequence which is the same as the base sequence of SEQ ID NO:38 except that not more than 4 bases are substituted at a genetic polymorphism site(s) therein. Specific examples of this set include the set of LMO2736-F222 and LMO2736-R591 in the Examples described below.

(D-2) A set of a mixed forward primer containing in its 3′-side a sequence having not less than 15 consecutive bases, preferably not less than 18 consecutive bases in the base sequence of SEQ ID NO:70, more preferably the full-length sequence of the base sequence, and a mixed reverse primer containing in its 3″-side a sequence having not less than 15 consecutive bases, preferably not less than 18 consecutive bases in the base sequence of SEQ ID NO:74, more preferably the full-length sequence of the base sequence. Specific examples of this set include the set of LMO2736-F222/M and LMO2736-R591/M in the Examples described below.

(E-1) A set of a forward primer containing in its 3′-side a sequence having not less than 15 consecutive bases, preferably not less than 18 consecutive bases in the base sequence of SEQ ID NO:34, more preferably the full-length sequence of the base sequence, or a sequence which is the same as the base sequence of SEQ ID NO:34 except that not more than 4 bases are substituted at a genetic polymorphism site(s) therein, and a reverse primer containing in its 3′-side a sequence having not less than 15 consecutive bases, preferably not less than 18 consecutive bases in the base sequence of SEQ ID NO:38, more preferably the full-length sequence of the base sequence, or a sequence which is the same as the base sequence of SEQ ID NO:38 except that not more than 4 bases are substituted at a genetic polymorphism site(s) therein. Specific examples of this set include the set of LMO2736-F488 and LMO2736-R591 in the Examples described below.

(E-2) A set of a mixed forward primer containing in its 3′-side a sequence having not less than 15 consecutive bases, preferably not less than 18 consecutive bases in the base sequence of SEQ ID NO:71, more preferably the full-length sequence of the base sequence, and a mixed reverse primer containing in its 3′-side a sequence having not less than 15 consecutive bases, preferably not less than 18 consecutive bases in the base sequence of SEQ ID NO:74, more preferably the full-length sequence of the base sequence. Specific examples of this set include the set of LMO2736-F488/M and LMO2736-R591/M in the Examples described below.

(F-1) A set of a forward primer containing in its 3′-side a sequence having not less than 15 consecutive bases, preferably not less than 18 consecutive bases in the base sequence of SEQ ID NO:34, more preferably the full-length sequence of the base sequence, or a sequence which is the same as the base sequence of SEQ ID NO:34 except that not more than 4 bases are substituted at a genetic polymorphism site(s) therein, and a reverse primer containing in its 3′-side a sequence having not less than 15 consecutive bases, preferably not less than 18 consecutive bases in the base sequence of SEQ ID NO:39, more preferably the full-length sequence of the base sequence, or a sequence which is the same as the base sequence of SEQ ID NO:39 except that not more than 4 bases are substituted at a genetic polymorphism site(s) therein. Specific examples of this set include the set of LMO2736-F488 and LMO2736-R685 in the Examples described below.

(F-2) A set of a mixed forward primer containing in its 3′-side a sequence having not less than 15 consecutive bases, preferably not less than 18 consecutive bases in the base sequence of SEQ ID NO:71, more preferably the full-length sequence of the base sequence, and a mixed reverse primer containing in its 3′-side a sequence having not less than 15 consecutive bases, preferably not less than 18 consecutive bases in the base sequence of SEQ ID NO:75, more preferably the full-length sequence of the base sequence. Specific examples of this set include the set of LMO2736-F488/M and LMO2736-R685/M in the Examples described below.

(G-1) A set of a forward primer containing in its 3′-side a sequence having not less than 15 consecutive bases, preferably not less than 18 consecutive bases in the base sequence of SEQ ID NO:34, more preferably the full-length sequence of the base sequence, or a sequence which is the same as the base sequence of SEQ ID NO:34 except that not more than 4 bases are substituted at a genetic polymorphism site(s) therein, and a reverse primer containing in its 3′-side a sequence having not less than 15 consecutive bases, preferably not less than 18 consecutive bases in the base sequence of SEQ ID NO:40, more preferably the full-length sequence of the base sequence, or a sequence which is the same as the base sequence of SEQ ID NO:40 except that not more than 4 bases are substituted at a genetic polymorphism site(s) therein. Specific examples of this set include the set of LMO2736-F488 and LMO2736-R771 in the Examples described below.

(G-2) A set of a mixed forward primer containing in its 3′-side a sequence having not less than 15 consecutive bases, preferably not less than 18 consecutive bases in the base sequence of SEQ ID NO:71, more preferably the full-length sequence of the base sequence, and a mixed reverse primer containing in its 3′-side a sequence having not less than 15 consecutive bases, preferably not less than 18 consecutive bases in the base sequence of SEQ ID NO:76, more preferably the full-length sequence of the base sequence. Specific examples of this set include the set of LMO2736-F488/M and LMO2736-R771/M in the Examples described below.

(H-1) A set of a forward primer containing in its 3′-side a sequence having not less than 15 consecutive bases, preferably not less than 18 consecutive bases in the base sequence of SEQ ID NO:35, more preferably the full-length sequence of the base sequence, or a sequence which is the same as the base sequence of SEQ ID NO:35 except that not more than 4 bases are substituted at a genetic polymorphism site(s) therein, and a reverse primer containing in its 3′-side a sequence having not less than 15 consecutive bases, preferably not less than 18 consecutive bases in the base sequence of SEQ ID NO:39, more preferably the full-length sequence of the base sequence, or a sequence which is the same as the base sequence of SEQ ID NO:39 except that not more than 4 bases are substituted at a genetic polymorphism site(s) therein. Specific examples of this set include the set of LMO2736-F530 and LMO2736-R685 in the Examples described below.

(H-2) A set of a mixed forward primer containing in its 3′-side a sequence having not less than 15 consecutive bases, preferably not less than 18 consecutive bases in the base sequence of SEQ ID NO:72, more preferably the full-length sequence of the base sequence, and a mixed reverse primer containing in its 3′-side a sequence having not less than 15 consecutive bases, preferably not less than 18 consecutive bases in the base sequence of SEQ ID NO:75, more preferably the full-length sequence of the base sequence. Specific examples of this set include the set of LMO2736-F530/M and LMO2736-R685/M in the Examples described below.

(I-1) A set of a forward primer containing in its 3″-side a sequence having not less than 15 consecutive bases, preferably not less than 18 consecutive bases in the base sequence of SEQ ID NO:35, more preferably the full-length sequence of the base sequence, or a sequence which is the same as the base sequence of SEQ ID NO:35 except that not more than 4 bases are substituted at a genetic polymorphism site(s) therein, and a reverse primer containing in its 3′-side a sequence having not less than 15 consecutive bases, preferably not less than 18 consecutive bases in the base sequence of SEQ ID NO:40, more preferably the full-length sequence of the base sequence, or a sequence which is the same as the base sequence of SEQ ID NO:40 except that not more than 4 bases are substituted at a genetic polymorphism site(s) therein. Specific examples of this set include the set of LMO2736-F530 and LMO2736-R771 in the Examples described below.

(I-2) A set of a forward primer containing in its 3′-side a sequence having not less than 15 consecutive bases, preferably not less than 18 consecutive bases in the base sequence of SEQ ID NO:60, more preferably the full-length sequence of the base sequence, or a sequence which is the same as the base sequence of SEQ ID NO:60 except that not more than 4 bases are substituted at a genetic polymorphism site(s) therein, and a reverse primer containing in its 3′-side a sequence having not less than 15 consecutive bases in the base sequence of SEQ ID NO:61, preferably the full-length sequence of the base sequence, or a sequence which is the same as the base sequence of SEQ ID NO:61 except that not more than 4 bases are substituted at a genetic polymorphism site(s) therein. Specific examples of this set include the set of the LAMP primers LMO2736-1 FIP and LMO2736-1 BIP in the Examples described below.

(I-3) A set of a forward primer containing in its 3′-side a sequence having not less than 15 consecutive bases, preferably not less than 18 consecutive bases in the base sequence of SEQ ID NO:62, more preferably the full-length sequence of the base sequence, or a sequence which is the same as the base sequence of SEQ ID NO:62 except that not more than 4 bases are substituted at a genetic polymorphism site(s) therein, and a reverse primer containing in its 3′-side a sequence having not less than 15 consecutive bases in the base sequence of SEQ ID NO:61, preferably the full-length sequence of the base sequence, or a sequence which is the same as the base sequence of SEQ ID NO:61 except that not more than 4 bases are substituted at a genetic polymorphism site(s) therein. Specific examples of this set include the set of the LAMP primers LMO2736-2 FIP and LMO2736-2 BIP in the Examples described below.

(I-4) A set of a mixed forward primer containing in its 3′-side a sequence having not less than 15 consecutive bases, preferably not less than 18 consecutive bases in the base sequence of SEQ ID NO:72, more preferably the full-length sequence of the base sequence, and a mixed reverse primer containing in its 3′-side a sequence having not less than 15 consecutive bases, preferably not less than 18 consecutive bases in the base sequence of SEQ ID NO:76, more preferably the full-length sequence of the base sequence. Specific examples of this set include the set of LMO2736-F530/M and LMO2736-R771/M in the Examples described below.

(J-1) A set of a forward primer containing in its 3′-side a sequence having not less than 15 consecutive bases, preferably not less than 18 consecutive bases in the base sequence of SEQ ID NO:35, more preferably the full-length sequence of the base sequence, or a sequence which is the same as the base sequence of SEQ ID NO:35 except that not more than 4 bases are substituted at a genetic polymorphism site(s) therein, and a reverse primer containing in its 3′-side a sequence having not less than 15 consecutive bases, preferably not less than 18 consecutive bases in the base sequence of SEQ ID NO:41, more preferably the full-length sequence of the base sequence, or a sequence which is the same as the base sequence of SEQ ID NO:41 except that not more than 4 bases are substituted at a genetic polymorphism site(s) therein. Specific examples of this set include the set of LMO2736-F530 and LMO2736-R992 in the Examples described below.

(J-2) A set of a mixed forward primer containing in its 3′-side a sequence having not less than 15 consecutive bases, preferably not less than 18 consecutive bases in the base sequence of SEQ ID NO:72, more preferably the full-length sequence of the base sequence, and a reverse primer containing in its 3′-side a sequence having not less than 15 consecutive bases, preferably not less than 18 consecutive bases in the base sequence of SEQ ID NO:41, more preferably the full-length sequence of the base sequence, or a sequence which is the same as the base sequence of SEQ ID NO:41 except that not more than 4 bases are substituted at a genetic polymorphism site(s) therein. Specific examples of this set include the set of LMO2736-F530/M and LMO2736-R992 in the Examples described below.

(K-1) A set of a forward primer containing in its 3′-side a sequence having not less than 15 consecutive bases, preferably not less than 18 consecutive bases in the base sequence of SEQ ID NO:36, more preferably the full-length sequence of the base sequence, or a sequence which is the same as the base sequence of SEQ ID NO:36 except that not more than 4 bases are substituted at a genetic polymorphism site(s) therein, and a reverse primer containing in its 3′-side a sequence having not less than 15 consecutive bases, preferably not less than 18 consecutive bases in the base sequence of SEQ ID NO:39, more preferably the full-length sequence of the base sequence, or a sequence which is the same as the base sequence of SEQ ID NO:39 except that not more than 4 bases are substituted at a genetic polymorphism site(s) therein. Specific examples of this set include the set of LMO2736-F572 and LMO2736-R685 in the Examples described below

(K-2) A set of a mixed forward primer containing in its 3′-side a sequence having not less than 15 consecutive bases, preferably not less than 18 consecutive bases in the base sequence of SEQ ID NO:73, more preferably the full-length sequence of the base sequence, and a mixed reverse primer containing in its 3′-side a sequence having not less than 15 consecutive bases, preferably not less than 18 consecutive bases in the base sequence of SEQ ID NO:75, more preferably the full-length sequence of the base sequence. Specific examples of this set include the set of LMO2736-F572/M and LMO2736-R685/M in the Examples described below.

(L-1) A set of a forward primer containing in its 3′-side a sequence having not less than 15 consecutive bases, preferably not less than 18 consecutive bases in the base sequence of SEQ ID NO:36, more preferably the full-length sequence of the base sequence, or a sequence which is the same as the base sequence of SEQ ID NO:36 except that not more than 4 bases are substituted at a genetic polymorphism site(s) therein, and a reverse primer containing in its 3′-side a sequence having not less than 15 consecutive bases, preferably not less than 18 consecutive bases in the base sequence of SEQ ID NO:40, more preferably the full-length sequence of the base sequence, or a sequence which is the same as the base sequence of SEQ ID NO:40 except that not more than 4 bases are substituted at a genetic polymorphism site(s) therein. Specific examples of this set include the set of LMO2736-F572 and LMO2736-R771 in the Examples described below.

(L-2) A set of a mixed forward primer containing in its 3′-side a sequence having not less than 15 consecutive bases, preferably not less than 18 consecutive bases in the base sequence of SEQ ID NO:73, more preferably the full-length sequence of the base sequence, and a mixed reverse primer containing in its 3′-side a sequence having not less than 15 consecutive bases, preferably not less than 18 consecutive bases in the base sequence of SEQ ID NO:76, more preferably the full-length sequence of the base sequence. Specific examples of this set include the set of LMO2736-F572/M and LMO2736-R771/M in the Examples described below.

(M-1) A set of a forward primer containing in its 3′-side a sequence having not less than 15 consecutive bases, preferably not less than 18 consecutive bases, more preferably not less than 20 consecutive bases in the base sequence of SEQ ID NO:63, still more preferably the full-length sequence of the base sequence, or a sequence which is the same as the base sequence of SEQ ID NO:63 except that not more than 4 bases are substituted at a genetic polymorphism site(s) therein, and a reverse primer containing in its 3′-side the base sequence of SEQ ID NO:64 or a sequence which is the same as the base sequence of SEQ ID NO:64 except that not more than 4 bases are substituted at a genetic polymorphism site(s) therein. Specific examples of this set include the set of the LAMP primers LMO2736-10 FIP and LMO2736-10 BIP in the Examples described below.

(N-1) A set of a mixed forward primer containing in its 3′-side a sequence having not less than 15 consecutive bases, preferably not less than 18 consecutive bases in the base sequence of SEQ ID NO:71, more preferably the full-length sequence of the base sequence, and a reverse primer containing in its 3′-side a sequence having not less than 15 consecutive bases, preferably not less than 18 consecutive bases in the base sequence of SEQ ID NO:41, more preferably the full-length sequence of the base sequence, or a sequence which is the same as the base sequence of SEQ ID NO:41 except that not more than 4 bases are substituted at a genetic polymorphism site(s) therein. Specific examples of this set include the set of LMO2736-F488/M and LMO2736-R992 in the Examples described below.

(O-1) A set of a mixed forward primer containing in its 3′-side a sequence having not less than 15 consecutive bases, preferably not less than 18 consecutive bases in the base sequence of SEQ ID NO:73, more preferably the full-length sequence of the base sequence, and a reverse primer containing in its 3′-side a sequence having not less than 15 consecutive bases, preferably not less than 18 consecutive bases in the base sequence of SEQ ID NO:41, more preferably the full-length sequence of the base sequence, or a sequence which is the same as the base sequence of SEQ ID NO:41 except that not more than 4 bases are substituted at a genetic polymorphism site(s) therein. Specific examples of this set include the set of LMO2736-F572/M and LMO2736-R992 in the Examples described below.

A primer containing a particular sequence in its 3′-side includes a primer in which an arbitrary sequence is added to the 5′-side of the particular sequence, and a primer composed of the particular sequence. For example, a primer containing the base sequence of SEQ ID NO:26 in its 3′-side includes a primer in which an arbitrary sequence is added to the 5′-side of the base sequence of SEQ ID NO:26, and a primer composed of the base sequence of SEQ ID NO:26.

Preferred specific examples of the genetic polymorphism sites in the base sequences described in (A-1) to (0-1) are as follows. Preferred specific examples of the primers containing a base substitution include primers each containing in its 3′-side a sequence in which at least one base selected from the following specific examples of genetic polymorphism sites is substituted. These specific examples are genetic polymorphism sites specified based on an alignment of the 12 kinds of lmo0084 gene sequences of 12 serotypes of SEQ ID NOs:1 to 12, and an alignment of the 13 kinds of lmo2736 gene sequences of 12 serotypes of SEQ ID NOs:13 to 25. It should noted, however, that genetic polymorphism sites other than the following specific examples may be found in cases where gene sequences of monocytogenes bacterial strains of other serotypes or other monocytogenes bacterial strains of the same serotypes are further taken into account, and that base substitutions in such sites are acceptable in the present invention. Thus, the genetic polymorphism sites in the sequences in the present invention are not limited to the following specific examples.

SEQ ID NO:26: position 6, position 15, and position 16

SEQ ID NO:27: position 6, position 15, and position 16

SEQ ID NO:28: position 2, position 11, and position 20

SEQ ID NO:29: position 2, position 11, and position 20

SEQ ID NO:30: position 8 and position 11

SEQ ID NO:31: position 8 and position 11

SEQ ID NO:33: position 5, position 18, and position 20

SEQ ID NO:34: position 5 and position 8

SEQ ID NO:35: position 5 and position 11

SEQ ID NO:36: position 5 and position 11

SEQ ID NO:38: position 10 and position 16

SEQ ID NO:39: position 14, position 15, and position 16

SEQ ID NO:40: position 7

SEQ ID NO:58: position 5, position 9, position 11, and position 14

SEQ ID NO:59: position 1 and position 10

SEQ ID NO:60: position 7 and position 13

SEQ ID NO:61: position 5

SEQ ID NO:62: position 7 and position 13

SEQ ID NO:63: position 1, position 4, position 19, and position 25

SEQ ID NO:64: position 6 and position 15

Preferred specific examples of the arbitrary additional sequence that may be present in the 5′-side of the primer include an additional sequence for construction of a LAMP primer. By selecting an arbitrary partial region positioned in the inner side relative to the target region of the primer, and adding the complementary strand of the partial region to the 5′-side of the primer, a LAMP primer can be constructed. Software for designing LAMP primers is known, and such known software can be used for designing LAMP primers for specific detection of the monocytogenes bacterium based on the specific primer setting regions (1) to (14) described above.

In the designing of a LAMP primer, the regions F3, F2, F1, B1, B2, and B3, located in this order from the 5′-upstream side, are necessary. A LAMP primer set is constituted with an FIP primer, in which the complementary sequence (the sequence of the antisense strand) of the F1 sequence is added to the 5′-end of F2; a BIP primer, in which the complementary sequence (the sequence of the sense strand) of the B1 sequence is added to the 5′-end of B2; a forward primer that hybridizes to the F3 region, and a reverse primer that hybridizes to the B3 region. The specific primer setting regions (1) to (14) described above may be employed for at least one of F2 and B2 among these, preferably for both of these. When the design is based on the primer sets of (a) to (v), in cases where the amplification size of the set is about 200 to 300 bp, both F2 and B2 may be selected such that they overlap with the primer setting regions. In cases where the amplification size of the set is outside this range, one of F2 and B2 may be selected such that it overlaps with the primer setting regions, and the other may be appropriately selected from candidate sequences proposed by the software.

The following (i) to (iv) are LAMP primer sets each of which was designed based on the set of LMO0084-F286A and LMO0084-R757B, which is one example of the primer set of (A-2), and the set of LMO2736-F530 and LMO2736-R771, which is one example of the primer set of (I-1). Preferred specific examples of the LAMP primer set for detection of the monocytogenes bacterium include these sets.

(i) A set of an F3 primer composed of the base sequence of SEQ ID NO:42, a B3 primer composed of the base sequence of SEQ ID NO:43, an FIP primer composed of the base sequence of SEQ ID NO:44, and a BIP primer composed of the base sequence of SEQ ID NO:45.

(ii) A set of an F3 primer composed of the base sequence of SEQ ID NO:46, a B3 primer composed of the base sequence of SEQ ID NO:47, an HP primer composed of the base sequence of SEQ ID NO:48, and a BIP primer composed of the base sequence of SEQ ID NO:49.

(iii) A set of an F3 primer composed of the base sequence of SEQ ID NO:50, a B3 primer composed of the base sequence of SEQ ID NO:51, an FIP primer composed of the base sequence of SEQ ID NO:52, and a BIP primer composed of the base sequence of SEQ ID NO:53.

(iv) A set of an F3 primer composed of the base sequence of SEQ ID NO:54, a B3 primer composed of the base sequence of SEQ ID NO:55, an HP primer composed of the base sequence of SEQ ID NO:56, and a BIP primer composed of the base sequence of SEQ ID NO:57.

Isothermal amplification primers used of methods other than the LAMP method may also be designed using known software or the like based on the specific primer setting regions (1) to (14) described above.

Preferred specific examples of the probe for detection of the PCR amplification product include probes containing oligonucleotide portions having the following sequences. The probe containing an oligonucleotide portion having the sequence of SEQ ID NO:85 is a mixed probe of a probe containing an oligonucleotide portion in which n---n is T---A (SEQ ID NO:78), and a probe containing an oligonucleotide portion in which n---n is C---C(SEQ ID NO:79). Similarly, the probe containing an oligonucleotide portion having the sequence of SEQ ID NO:86 is a mixed probe of a probe containing an oligonucleotide portion in which n----n is G----G (SEQ ID NO:83), and a probe containing an oligonucleotide portion in which n----n is C----C(SEQ ID NO:84).

<Probes for LMO0084 Gene>

(SEQ ID NO: 77)

TATTACATTCATAGAATTGACCC (set at position 366 to

position 389)

(SEQ ID NO: 85)

ATCTGGTGGCGAGAAGCnGAAnA (set at position 535 to

position 558; nGAAn is TGAAA or CGAAC)

(SEQ ID NO: 80)

TACCAAGATTCCAAAAAGAAGCCATG (set at position 686 to

position 711)

<Probes for LMO2736 Gene>

(SEQ ID NO: 81)

AAAAAAGGCTGGACTAAAGC (set at position 70 to

position 89)

(SEQ ID NO: 82)

ACGTCAAAAAAATCATTATC (set at position 372 to

position 393)

(SEQ ID NO: 86)

GTTTTCGGTGCTCAAAAAGGnGCAAnTCC (set at position

619 to position 647; nGCAAn is GGCAAG or CGCAAC)

Each of the probe of SEQ ID NO:85 and the probe of SEQ ID NO:86 is a mixed probe of two kinds of oligonucleotide probes. The mixing ratio of these two kinds of probes, in terms of the molar ratio, may be about 1:5 to 5:1, for example, about 1:2 to 2:1, or about 1:1.5 to 1.5:1. The probes can be preferably used at a mixing ratio of 1:1.

Since the position where each probe is set is as described above, the probe may be used in combination with a primer set which amplifies a region containing this set region. A probe containing an oligonucleotide portion having these sequences can be preferably used as a capture probe for nucleic acid chromatography or a probe for real-time PCR. In cases where the probe is used as a real-time PCR probe, the 5′-end and the 3′-end of the oligonucleotide may be modified with a fluorescent substance and a quencher substance. It is common to modify the 5′-end with a fluorescent substance, and the 3′-end with a quencher substance. Especially preferred combinations of the primers and the probe are described in Table 23 and Table 25 in the following Examples.

Examples

The present invention is described below more concretely by way of Examples. However, the present invention is not limited to the following Examples.

I. Search for Target Genes

Conventional products for gene testing of the monocytogenes bacterium target pathogenicity genes of the monocytogenes bacterium, such as the hlyA gene, clpC gene, inlA gene, and plcA gene. However, they are not capable of distinguishing the monocytogenes bacterium from other bacteria belonging to the genus Listeria. Aiming at establishment of a primer set capable of distinguishing the monocytogenes bacterium from other bacteria belonging to the genus Listeria with high accuracy, a study was carried out using genes other than the pathogenicity genes described above as targets.

First, the site of http://genolist.pasteur.fr/ListiList/ was used. The information on monocytogenes in Accession No. NC_003210.1 and innocua in Accession No. NC_003212.1 on this site was utilized. Listeria innocua (number of genes: 3068) and Listeria monocytogenes (number of genes: 2941), which belong to the genus Listeria, were subjected to comparative genomic analysis to narrow down monocytogenes-specific genes to 296 genes.

Subsequently, for each of the selected 296 genes, BLAST search was carried out against a database to investigate whether or not the gene can be confirmed to be present in the genome sequences of all isolated strains of monocytogenes of each serotype deposited therein. Genes whose presence could not be confirmed in any of the isolated strains were excluded from the candidates. Examples of the search results are shown in Table 3.

TABLE 3

Serotype
LM000038
LM000077
LM000313
LMO02387
LMO02736

L. monocytogenes

1/2a
18
18
1
18
18

L. monocytogenes

1/2b
5
5
2
5
5

L. monocytogenes

1/2c
2
2
2
2
2

L. monocytogenes

3a
2
2
0
2
2

L. monocytogenes

3b
1
1
0
1
1

L. monocytogenes

3c
1
1
1
1
1

L. monocytogenes

4a
0
3
0
3
3

L. monocytogenes

4b
12
12
5
12
12

L. monocytogenes

4c
1
1
0
1
1

L. monocytogenes

4d
1
1
0
1
1

L. monocytogenes

4e
0
0
0
1
1

L. monocytogenes

7
1
1
0
1
1

There is/are
There is/are
There is/are
The
The

an isolated
an isolated
an isolated
presence of
presence of

strain(s) for
strain(s) for
strain(s) for
the gene
the gene

which the
which the
which the
could be
could be

presence of
presence of
presence of
confirmed
confirmed

the gene
the gene
the gene
in genomic
in genomic

cannot be
cannot be
cannot be
sequences
sequences

confirmed.
confirmed.
confirmed.
of all
of all

↓
↓
↓
isolated
isolated

Excluded
Excluded
Excluded
strains
strains

from
from
from
deposited.
deposited.

candidates
candidates
candidates
↓
↓

Selected as
Selected as

a candidate
a candidate

By this, the candidate genes were finally narrowed down to 6 genes (LMO 0083, LMO 0084, LMO 0444, LMO 0833, LMO 2387, and LMO 2736).

For each of the 6 genes, a plurality of PCR primers were designed, and PCR was actually carried out for the 6 strains of the monocytogenes bacterium (serotypes 1/2a, 1/2b, 1/2c, 4a, 4b, and 4d) and 3 strains of other bacteria belonging to the genus Listeria (L. innocua, L. grayi, and L. ivanovii), to study specificity to the monocytogenes bacterium. Based on comparison among sequences of various serotypes of monocytogenes (using the sequences of the accession numbers described above in Table 2), the PCR primers in this study were designed such that they target common regions. As a result, with LMO 0083, LMO 0444, LMO 0833, and MLO 2387, detection of some of the 6 strains was unsuccessful, or amplification occurred with other bacteria belonging to the genus Listeria. Thus, design of a primer set having high specificity was difficult therewith. For example, in the case of the LMO0833 gene, specificity was obtained since no amplification of the bacteria belonging to the genus Listeria was found as a result of combination of the primer F329 (ggaaagcaattgtccactcga; SEQ ID NO:65) and the primer R610 (tgttggtgagtagcgtggaa; SEQ ID NO:66). However, monocytogenes of the serotype 4a also did not show the amplification. Table 4 shows examples of the PCR results for the candidate genes. With LMO 0084 and LMO 2736, specific amplification products were obtained only from the 6 strains of the monocytogenes bacterium. For comparison, two commercially available kits for gene testing of the monocytogenes bacterium were used for detection of the same bacterial strains. As a result, neither of these succeeded in specific detection of the monocytogenes bacteria used herein (Table 4). From these results, the candidate genes were narrowed down to LMO 0084 and LMO 2736, and construction of monocytogenes bacterium-specific primers was attempted therewith.

TABLE 4

L. monocytogenes

L. monocytogenes

L. monocytogenes

L. monocytogenes

L. monocytogenes

GTC02947
GTC02948
JCM7672
JCM7674
JCM7675

Gene name
Description
1/2a
1/2b
1/2C
4a
4b

LMO 0083
similar to transcription
−
+
+
+
+

regulator

LMO 0084
similar to oxidoreductases
+
+
+
+
+

LMO 0444
conserved hypothetical
+
+
+
−
−

protein

LMO 0833
similar to transcription
+
+
+
−
+

regulator

LMO 2387
conserved hypothetical
+
+
+
+
+

protein

LMO 2736
conserved hypothetical
+
+
+
+
+

protein

hlyA gene
Pathogenic Bacterial
+
+
+
−
−

Multiplex PCR Detection kit

TA10 (TAKARA: RR106A)

(Unknown)
mericon L. monocytogenes
+
+
+
+
+

Kit (QIAGEN: 290023)

L. monocytogenes

L. gayi

L. innocua

L. Ivaimii

JCM7680
GTC02964T
GTC16426T
JCM7681

Gene name
Description
4d
−
−
−

LMO 0083
similar to transcription
+
−
−
−

regulator

LMO 0084
similar to oxidoreductases
+
−
−

LMO 0444
conserved hypothetical
−
−
−
−

protein

LMO 0833
similar to transcription
+
−
−
−

regulator

LMO 2387
conserved hypothetical
+
+
−
+

protein

LMO 2736
conserved hypothetical
+
−
−
−

protein

hlyA gene
Pathogenic Bacterial
−
−
−
−

Multiplex PCR Detection kit

TA10 (TAKARA: RR106A)

(Unknown)
mericon L. monocytogenes
+
+
+
+

Kit (QIAGEN: 290023)

II. Construction of Monocytogenes Bacterium-Specific Primer Sets

Base sequences of the two genes LMO 0084 and LMO 2736 in various serotypes, identified by the narrowing down as described above, were studied in more detail, and a large number of primers were designed therefrom. By performing a PCR study using an increased number of bacterial strains, construction of primers for specific detection of the monocytogenes bacterium with high accuracy was attempted.

<Methods>
1. Bacterial Strains Used

The bacterial strains subjected to the PCR test (Table 5-1 to Table 5-3) were obtained from Microbe Division, RIKEN BioResource Research Center (JCM); Center for Conservation of Microbial Genetic Resource, Organization for Research and Community Development, Gifu University (GTC); Department of Biotechnology, National Institute of Technology and Evaluation (IFO); JA Zen-noh Institute of Animal Health (JA); and Institute of Applied Microbiology, University of Tokyo (IMCB).

TABLE 5-1

Monocytogenes bacterium

Bacterial

strain No.
Microorganism name
Resource name
Serotype

1

L. monocytogenes

GTC02947
1/2a

2

L. monocytogenes

GTC02948
1/2b

3

L. monocytogenes

JCM7672
1/2c

4

L. monocytogenes

JCM7673
3a

5

L. monocytogenes

JCM7677
3b

6

L. monocytogenes

JCM7678
3c

7

L. monocytogenes

JCM7674
4a

8

L. monocytogenes

JCM7675
4b

9

L. monocytogenes

JCM7680
4d

10

L. monocytogenes

GTC02957
5

TABLE 5-2

Bacteria belonging to the genus Listeria

other than the monocytogenes bacterium

Bacterial

strain No.
Microorganism name
Resource name

11

L. ivanovii

GTC02961

12

L. ivanovii subsp. ivanovii
JCM7681

13

L. ivanovii subsp. ivanovii
GTC01640T

14

L. ivanovii subsp. londoniensis
GTC01641

15

L. innocua

GTC16426T

16

L. innocua

GTC02960

17

L. welshimeri

GTC02963T

18

L. seeligeri

GTC16428T

19

L. grayi

GTC02964T

20

L. murrayi

GTC02964

21

L. marthii

GTC16430T

22

L. rocourtiae

GTC16429T

TABLE 5-3

Food-poisoning bacteria other than Listeria bacteria

which tend to cause problems in the field of foods

Bacterial

strain No.
Microorganism name
Resource name
Serotype

23

Escherichia coli

ATCC10798

24

Salmonella subsp. enterica (I)
JA.107
Type I

25

Salmonella subsp. salamae (II)
JA.125
Type II

26

Salmonella subsp.
JA.76
Type IIIa

arizonae (IIIa)

27

Salmonella subsp.
JA.129
Type IIIb

diarizinae (IIIb)

28

Salmonella subsp. houtenae (IV)
JA.n-22
Type IV

29

Salmonella bongori (V)
JA.94
Type V

30

Salmonella subsp.
ATCC43971

enterica Typhimurium

31

Staphylococcus aureus

ATCC6538P

32

Staphylococcus aureus

ATCC25923

33

Staphylococcus aureus

ATCC29213

34

Staphylococcus aureus

JMC2197

35

Staphylococcus aureus

IMCB.IMA2

36

Staphylococcus cohnii

ATCC29974

37

Staphylococcus haemolyticus

ATCC29970

38

Staphylococcus hyicus subsp.
ATCC11249

39

Staphylococcus intermedius

ATCC29663

40

Staphylococcus saprophyticus

ATCC15305

41

Citrobacter freundii

ATCC8090

42

Citrobacter freundii

ATCC8043

43

Proteus vulgaris

IFO3988

44

Lactobacillus bulgaricus

IFO13953

45

Lactobacillus helveticus

IFO3809

46

Streptococcus sp.
IFO3535

47

Streptococcus sanguis

ATCC10558

48

Streptococcus mitis

ATCC6249

2. Primers and PCR Reaction Conditions

Various primers were designed based on sequence information for the lmo0084 gene (SEQ ID NOs:1 to 12) and the lmo2736 gene (SEQ ID NOs:13 to 25) in various serotypes of Listeria monocytogenes. Table 6 shows part of those sequences. For the lmo0084 gene, primers of SEQ ID NOs:26, 28, and 30 were designed such that they reflect genetic polymorphism in the serotype 1/2a of the monocytogenes bacterium, and primers of SEQ ID NOs:27, 29, and 31 were designed such that they reflect genetic polymorphism in the serotype 4a. For the lmo2736 gene, primers of SEQ ID NOs:32, 37, and 41 were designed such that they reflect common sequences among the various serotypes of the monocytogenes bacterium, and primers of SEQ ID NOs:33, 34, 35, 36, 38, 39, and 40 were designed such that they reflect genetic polymorphism in the serotype 1/2c of the monocytogenes bacterium. The designed PCR primers were synthesized by custom synthesis by Fasmac Co., Ltd. Template DNA was obtained by extracting genomic DNA from each bacterial strain using a mericon DNA Bacteria Plus Kit (QIAGEN).

TABLE 6

Oligonucleotide

SEQ ID

Target gene
name ^a)
Sequence ^b)
Setting position ^c)
NO.

lmo0084
LMO0084-F286A
AGCCGTCCAGAAAGCATCAA
286 to 305
26

-----*--------**----

LMO0084-F286B
AGCCGCCCAGAAAGTCTCAA
286 to 305
27

-----*--------**----

LMO0084-F281A
TCGATAGCCGTCCAGAAAGC
281 to 300
28

-*--------*--------*

LMO0084-F281B
TTGATAGCCGCCCAGAAAGT
281 to 300
29

-*--------*--------*

LMO0084-R757A
GCTCGTCGGCGATTTCTTTC
738 to 757
30

-------*--*---------

LMO0084-R757B
GCTCGTCGGCTATTTCTTTC
738 to 757
31

-------*--*---------

lmo2736
LMO2736-F8
TCGTCATCGCACCTGATTCA
8 to 27
32

--------------------

LMO2736-F222
GGCCTCCTACGGTATTCACG
222 to 241
33

----*------------*-*

LMO2736-F488
CCGGTGGCATTCATTTGCAA
488 to 507
34

----*--*------------

LMO2736-F530
GCAACCTTAACCCAAAGCTG
530 to 549
35

----*-----*---------

LMO2736-F572
CCTGTGACGTSACGAATCCA
572 to 591
36

----*-----*---------

LMO2736-R176
TCCACCTCGGAAGACTCACT
157 to 176
37

--------------------

LMO2736-R591
TGGATTCGTCACGTCACAGG
572 to 591
38

---------*-----*----

LMO2736-R685
AGTTCTGCATGGCGTTCTCT
666 to 685
39

-------------***----

LMO2736-R771
TAGTCCAGCAGCGATACCAC
752 to 771
40

------*-------------

LMO2736-R992
TTGTTTTCGAGTGCAAGGCT
973 to 992
41

--------------------

^a)In the oligonucleotide names, F represents “forward”, and R represents “reverse”.

^b)* represents a base showing polymorphism based on comparison among the sequences of SEQ ID NOs: 1 to 25.

^c)The setting position is described using as a standard SEQ ID NO: 1 in the cases of the primers targeting lmo0084, and SEQ ID NO: 13 in the cases of the primers targeting lmo2736.

The composition of the PCR reaction liquid is shown below in Table 7. The PCR was carried out using GeneAmp PCR System 9700. The reaction cycle was as follows: 94° C. for 2 minutes→(94° C. for 20 seconds→60° C. for 20 seconds→72° C. for 40 seconds)×30 cycles→72° C. for 7 minutes→4° C.

TABLE 7

Liquid

volume

Reagent
Manufacturer
Code. No.
(μL)

TaKaRa Ex Taq (5 U/μl)
TaKaRa
RR01AM
0.2

10 × Ex Taq Buffer (Mg²⁺ free)
TaKaRa
RR01AM
2.0

MgCl₂(25 mM)
TaKaRa
RR01AM
1.6

dNTP Mixture (2.5 mM each)
TaKaRa
RR01AM
1.6

100 μM Primer F
Fasmac
—
0.1

100 μM Primer R
Fasmac
—
0.1

D.W.
—
—
12.4

1 ng/μT Template DNA
—
—
2.0

Per tube

20.0

3. Selective Isolation Medium for Monocytogenes

Various bacteria belonging to the genus Listeria were plated on ALOA agar medium (Sysmex Corporation) or CHROMagar medium (Kanto Chemical Co., Inc.), and cultured at 37° C. for about 24 hours, followed by observation of colonies. The monocytogenes bacterium forms bluish-green colonies accompanied by milky-white halos on ALOA agar medium, and blue colonies accompanied by milky-white halos on CHROMagar medium.

In the halo formation test, all strains of the monocytogenes bacterium showed formation of halos to give positive results although some strains such as the bacterial strain No. 4 partially showed colonies forming no halo. On the other hand, L. ivanovii (bacterial strain Nos. 11, 12, 13, and 14) and L. seeligeri (bacterial strain No. 18) showed false-positive results. No colony formation was found for 26 food-poisoning bacterial strains other than those of the genus Listeria (bacterial strain Nos. 23 to 48). Part of the results of the halo test are shown in FIG. 2-1 to FIG. 2-3.

As a result of study using various combinations of the designed primers, the monocytogenes bacterium could be specifically detected with the combinations shown in Table 8-1 to Table 8-4 independent of genetic polymorphism. None of these combinations produced a PCR product having the specific size from bacteria belonging to the genus Listeria other than the monocytogenes bacterium (bacterial strain Nos. 11 to 22), or from the other 26 food-poisoning bacterial strains (bacterial strain Nos. 23 to 48) (Table 9-1 to Table 9-6). Examples of the PCR results are shown in FIG. 2-1 and FIG. 2-2.

Since L. ivanovii and L. seeligeri form halos similarly to the monocytogenes bacterium on ALOA agar medium and CHIROMagar medium, which are commonly used for selective isolation of the monocytogenes bacterium, they cannot be easily distinguished from the monocytogenes bacterium. However, with the primer sets shown in Table 8-1 to Table 8-4, various isolated bacterial strains of these bacteria belonging to the genus Listeria showed no amplification, giving negative results. On the other hand, the monocytogenes bacterial strain JMC7673 (bacterial strain No. 4 in the tables) could also be detected as the monocytogenes bacterium in spite of the fact that it also produces colonies forming no halo. Thus, it could be confirmed that the primer sets shown in Table 8-1 to Table 8-4 have very high specificities to the monocytogenes bacterium. It could be further confirmed that those primer sets are superior to the conventional monocytogenes bacterium detection PCR kits shown in Table 3.

TABLE 8-1

PCR for detection of the lmo0084 gene, and halo formation

primer

SEQ

SEQ
Amplification
Bacterial strain No. (monocytogenes bacterium)

set No.
F primer
ID NO.
R primer
ID NO.
size (bp)
1
2
3
4
5
6
7
8
9
10

6
F286A
26
R757A
30
471
+
+
+
+
+
+
+
+
+
+

8
F286A
26
R757B
31
471
+
+
+
+
+
+
+
+
+
+

14
F286B
27
R757A
30
471
+
+
+
+
+
+
+
+
+
+

16
F286B
27
R757B
31
471
+
+
+
+
+
+
+
+
+
+

2
F281A
28
R757A
30
476
+
+
+
+
+
+
+
+
+
+

4
F281A
28
R757B
31
476
+
+
+
+
+
+
+
+
+
+

10
F281B
29
R757A
30
476
+
+
+
+
+
+
+
+
+
+

12
F281B
29
R757B
31
476
+
+
+
+
+
+
+
+
+
+

Halo formation
(+)
(+)
(+)
(+/−)
(+)
(+)
(+)
(+)
(+)
(+)

TABLE 8-2

PCR for detection of the lmo0084 gene, and halo formation

Bacterial strain No. (bacteria belonging to the genus

primer

Amplification

Listeria other than the monocytogenes bacterium)

set No.
F primer
R primer
size (bp)
11
12
13
14
15
16
17
18
19
20
21
22

6
F286A
R7C7A
471
−
−
−
−
−
−
−
−
−
−
−
−

8
F286A
R757B
471
−
−
−
−
−
−
−
−
−
−
−
−

14
F286B
R7S7A
471
−
−
−
−
−
−
−
−
−
−
−
−

16
F286B
R757B
471
−
−
−
−
−
−
−
−
−
−
−
−

2
F281A
R757A
476
−
−
−
−
−
−
−
−
−
−
−
−

4
F281A
R757B
476
−
−
−
−
−
−
−
−
−
−
−
−

10
F281B
R757A
476
−
−
−
−
−
−
−
−
−
−
−
−

12
F281B
R757B
476
−
−
−
−
−
−
−
−
−
−
−
−

Halo formation
(+)
(+)
(+)
(+)
(−)
(−)
(−)
(+)
(−)
(−)
(−)
(−)

TABLE 8-3

PCR for detection of the lmo2736 gene, and halo formation

primer

SEQ

SEQ
Amplification
Bacterial strain No. (monocytogenes bacterium)

set No.
F primer
ID NO.
R primer
ID NO.
size (bp)
1
2
3
4
5
6
7
8
9
10

1
F8
32
R176
37
168
+
+
+
+
+
+
+
+
+
+

3
F222
33
R591
38
369
+
+
+
+
+
+
+
+
+
+

4
F488
34
R591
38
103
+
+
+
+
+
+
+
+
+
+

5
F488
34
R685
39
197
+
+
+
+
+
+
+
+
+
+

6
F488
34
R771
40
283
+
+
+
+
+
+
+
+
+
+

9
F530
35
R685
39
155
+
+
+
+
+
+
+
+
+
+

10
F530
35
R771
40
241
+
+
+
+
+
+
+
+
+
+

12
F530
35
R992
41
462
+
+
+
+
+
+
+
+
+
+

13
F572
36
R685
39
113
+
+
+
+
+
+
+
+
+
+

14
F572
36
R771
40
199
+
+
+
+
+
+
+
+
+
+

Halo formation
(+)
(+)
(+)
(+/−)
(+)
(+)
(+)
(+)
(+)
(+)

TABLE 8-4

PGR for detection of the lmo2736 gene, and halo formation

Bacterial strain No. (bacteria belonging to the genus

primer

Amplification

Listeria other than the monocytogenes bacterium)

set No.
F primer
R primer
size (bp)
11
12
13
14
15
16
17
18
19
20
21
22

1
F8
R176
168
−
−
−
−
−
−
−
−
−
−
−
−

3
F222
R591
369
−
−
−
−
−
−
−
−
−
−
−
−

4
F488
R591
103
−
−
−
−
−
−
−
−
−
−
−
−

5
F488
R685
197
−
−
−
−
−
−
−
−
−
−
−
−

6
F488
R771
283
−
−
−
−
−
−
−
−
−
−
−
−

9
F530
R685
155
−
−
−
−
−
−
−
−
−
−
−
−

10
F530
R771
241
−
−
−
−
−
−
−
−
−
−
−
−

12
F530
R992
462
−
−
−
−
−
−
−
−
−
−
−
−

13
F572
R685
113
−
−
−
−
−
−
−
−
−
−
−
−

14
F572
R771
199
−
−
−
−
−
−
−
−
−
−
−
−

Halo formation
(+)
(+)
(+)
(+)
(−)
(−)
(−)
(+)
(−)
(−)
(−)
(−)

TABLE 9-1

24
25
26
27

Salmonella

Salmonella

Salmonella

Salmonella

23
subsp.
subsp.
subsp.
subsp.

Escherichia

enterica

salamae

arizonae

diariznae

LMO 00084

coli

(I)
(II)
(IIIa)
(IIIb)

No.
primer F
primer R
size
ATCC10798
JA.107
JA.125
JA.76
JA.129

6
286
A
757
A
471
−
−
−
−
−

8
286
A
757
B
471
−
−
−
−
−

14
286
B
757
A
471
−
−
−
−
−

16
286
B
757
B
471
−
−
−
−
−

2
281
A
757
A
476
−
−
−
−
−

4
281
A
757
B
476
−
−
−
−
−

10
281
B
757
A
476
−
−
−
−
−

12
281
B
757
B
476
−
−
−
−
−

28

30

Salmonella

29

Salmonella

subsp.

Salmonella

subsp.
31

houtenae

bongori

enterica

Staphylococcus

LMO 00084
(IV)
(V)

Typhimurium

aureus

No.
primer F
primer R
size
JA.n-22
JA.94
ATCC43971
ATCC6538P

6
286
A
757
A
471
−
−
−
−

8
286
A
757
B
471
−
−
−
−

14
286
B
757
A
471
−
−
−
−

16
286
B
757
B
471
−
−
−
−

2
281
A
757
A
476
−
−
−
−

4
281
A
757
B
476
−
−
−
−

10
281
B
757
A
476
−
−
−
−

12
281
B
757
B
476
−
−
−
−

TABLE 9-2

32
33
34
35
36

Staphylococcus

Staphylococcus

Staphylococcus

Staphylococcus

Staphylococcus

LMO 00084

aureus

aureus

aureus

aureus

cohnii

No.
primer F
primer R
size
ATCC25923
ATCC29213
JMC2197
IMCB.IMA2
ATCC29974

6
286
A
757
A
471
−
−
−
−
−

8
286
A
757
B
471
−
−
−
−
−

14
286
B
757
A
471
−
−
−
−
−

16
286
B
757
B
471
−
−
−
−
−

2
281
A
757
A
476
−
−
−
−
−

4
281
A
757
B
476
−
−
−
−
−

10
281
B
757
A
476
−
−
−
−
−

12
281
B
757
B
476
−
−
−
−
−

37
38
39
40

Staphylococcus

Staphylococcus

Staphylococcus

Staphylococcus

LMO 00084

haemolyticus

hyicus subsp.

intermedius

saprophyticus

No.
primer F
primer R
size
ATCC29970
ATCC11249
ATCC29663
ATCC15305

6
286
A
757
A
471
−
−
−
−

8
286
A
757
B
471
−
−
−
−

14
286
B
757
A
471
−
−
−
−

16
286
B
757
B
471
−
−
−
−

2
281
A
757
A
476
−
−
−
−

4
281
A
757
B
476
−
−
−
−

10
281
B
757
A
476
−
−
−
−

12
281
B
757
B
476
−
−
−
−

TABLE 9-3

41
42
43
44
45

Citrobacter

Cilrobacter

Proteus

Lactbacillus

Lactbacillus

LMO 00084

freundii

freundii

vulgaris

bulgarius

halveticus

No.
primer: F
primer: R
size
ATCC8090
ATCC8043
IFO3988
IFO13953
IFO3809

6
286
A
757
A
471
−
−
−
−
−

8
286
A
757
B
471
−
−
−
−
−

14
286
B
757
A
471
−
−
−
−
−

16
286
B
757
B
471
−
−
−
−
−

2
281
A
757
A
476
−
−
−
−
−

4
281
A
757
B
476
−
−
−
−
−

10
281
B
757
A
476
−
−
−
−
−

12
281
B
757
B
476
−
−
−
−
−

46
47
48

Streptcoccus

Streptcoccus

Streptcoccus

LMO 00084
sp.

sanguis

mitis

No.
primer: F
primer: R
size
IFO3535
ATCC10558
ATCC6249

6
286
A
757
A
471
−
−
−

8
286
A
757
B
471
−
−
−

14
286
B
757
A
471
−
−
−

16
286
B
757
B
471
−
−
−

2
281
A
757
A
476
−
−
−

4
281
A
757
B
476
−
−
−

10
281
B
757
A
476
−
−
−

12
281
B
757
B
476
−
−
−

TABLE 9-4

24
25
26
27
28

Salmonella

Salmonella

Salmonella

Salmonella

Salmonella

23
subsp.
subsp
subsp
subsp
subsp

Escherichia

enterica

salamae

arizonae

dianzinae

houtenae

LMO 02736

coli

(I)
(II)
(IIIa)
(IIIb)
(IV)

No
primer: F
printer: R
size
ATCC10798
JA.107
JA.125
JA.76
JA.129
JA.n-22

1
8
176
168
−
−
−
−
−
−

3
222
591
369
−
−
−
−
−
−

4
488
591
103
−
−
−
−
−
−

5
488
685
197
−
−
−
−
−
−

6
488
771
283
−
−
−
−
−
−

8
488
992
504
−
−
−
−
−
−

9
530
685
155
−
−
−
−
−
−

10
530
771
241
−
−
−
−
−
−

12
530
992
462
−
−
−
−
−
−

10
572
685
113
−
−
−
−
−
−

14
572
771
199
−
−
−
−
−
−

30

29

Salmonella

Salmonela

subsp
31

bongori

enterica

Staphylococcus

LMO 02736
(V)

Typhimurium

aureus

No
primer: F
printer: R
size
JA.94
ATCC43971
ATCC6538P

1
8
176
168
−
−
−

3
222
591
369
−
−
−

4
488
591
103
−
−
−

5
488
685
197
−
−
−

6
488
771
283
−
−
−

8
488
992
504
−
−
−

9
530
685
155
−
−
−

10
530
771
241
−
−
−

12
530
992
462
−
−
−

10
572
685
113
−
−
−

14
572
771
199
−
−
−

TABLE 9-5

32
33
34
35
36
37

Staphylococcus

Staphylococcus

Staphylococcus

Staphylococcus

Staphylococcus

Staphylococcus

LMO 02736

aurous

aureus

aureus

aureus

cohnii

haemotyticus

No
primer: F
primer: R
size
ATCC25923
ATCC29213
JMC2197
IMCB.IMA2
ATCC29974
ATCC29970

1
8
176
168
−
−
−
−
−
−

3
222
591
369
−
−
−
−
−
−

4
488
591
103
−
−
−
−
−
−

5
488
685
197
−
−
−
−
−
−

6
488
771
283
−
−
−
−
−
−

8
488
992
504
−
−
−
−
−
−

9
530
685
155
−
−
−
−
−
−

10
530
771
241
−
−
−
−
−
−

12
530
992
462
−
−
−
−
−
−

10
572
685
113
−
−
−
−
−
−

14
572
771
199
−
−
−
−
−
−

38
39
40

Staphylococcus

Staphylococcus

Staphylococcus

LMO 02736

hyicus subsp.

intermedius

saprophyticus

No
primer: F
primer: R
size
ATCC11249
ATCC29663
ATCC15305

1
8
176
168
−
−
−

3
222
591
369
−
−
−

4
488
591
103
−
−
−

5
488
685
197
−
−
−

6
488
771
283
−
−
−

8
488
992
504
−
−
−

9
530
685
155
−
−
−

10
530
771
241
−
−
−

12
530
992
462
−
−
−

10
572
685
113
−
−
−

14
572
771
199
−
−
−

TABLE 9-6

41
42
43
44
45

Citrobacter

Citrobacter

Proteus

Lactbacillus

Lactbacillus

LMO 02736

freundii

freundii

vulgaris

bulgarius

helveticus

No.
primer: F
primer: R
size
ATCC8090
ATCC8043
IFO3988
IFO13953
IFO3809

1
8
176
168
−
−
−
−
−

3
222
591
369
−
−
−
−
−

4
488
591
103
−
−
−
−
−

5
488
685
197
−
−
−
−
−

6
488
771
283
−
−
−
−
−

8
488
992
504
−
−
−
−
−

9
530
685
155
−
−
−
−
−

10
530
771
241
−
−
−
−
−

12
530
992
462
−
−
−
−
−

10
572
685
113
−
−
−
−
−

14
572
771
199
−
−
−
−
−

46
47
48

Streptcoccus

Streptcoccus

Streptcoccus

LMO 02736
sp.

sanguis

mitis

No.
primer: F
primer: R
size
IFO3535
ATCC10558
ATCC6249

1
8
176
168
−
−
−

3
222
591
369
−
−
−

4
488
591
103
−
−
−

5
488
685
197
−
−
−

6
488
771
283
−
−
−

8
488
992
504
−
−
−

9
530
685
155
−
−
−

10
530
771
241
−
−
−

12
530
992
462
−
−
−

10
572
685
113
−
−
−

14
572
771
199
−
−
−

LAMP primers were designed based on the primer set F286A/R757B, which targets the lmo0084 gene, and the primer set F530/R771, which targets the lmo2736 gene. For the designing of the primers, LAMP Designer 1.14 (manufactured by OptiGene Limited), which is known support software for designing primers for the LAMP method, was used.

[Designing of LAMP Method Primers Targeting lmo0084]

1. The search region was entered as 1 to 984.

2. The range from F2 to B2 was entered as 150 to 300.

3. Sequences were predicted for the sets of F3/B3, F2/B2, and F1/B1 by the software.

4. Sets were selected such that one of F2 and B2 overlaps with the PCR primer F286A or R757B.

5. Optimization was carried out to select sets in which both F2 and B2 sequences overlap with the primers F286A and R757B.

[Designing of LAMP Method Primers Targeting Lmo2736]

1. The search region was entered as 491 to 811.

2. The range from F2 to B2 was entered as 150 to 300.

3. The range from F1 to B1 was entered as 100 to 200.

4. Sequences were predicted for the sets of F3/B3, F2/B2, and F1/B1 by the software.

5. Sets were selected such that F2/B2 overlaps with the PCR primer F530 or R771.

6. The LAMP method was actually carried out with the designed primers, and optimization was carried out mainly for the F2/B2 selected.

The thus obtained LAMP primer sets for specific detection of the monocytogenes bacterium are shown below. The lmo0084 LAMP primer set was designed such that it reflects the genetic polymorphism in the serotype 1/2a of the monocytogenes bacterium. The lmo2736 LAMP primer sets were designed such that they reflect the genetic polymorphism in the serotype 1/2c of the monocytogenes bacterium except for SEQ ID NO:64. As a result of detection tests using the above bacterial strains, all of the primer sets were found to have specificity to the monocytogenes bacterium without being influenced by the genetic polymorphisms, as shown below in Table 14-1 to Table 14-3.

TABLE 10

lmo0084 LAMP primer set

SEQ

Sequence (5′ → 3′)
ID No.
Setting position

LMO84 F3
AAATGATTGAAGTCGTACGC
42
104-123

LMO84 B3
GCAACCTCTTCAATTGGGATA
43
384-404

LMO84 FIP
CTAAAGCTTCTCCGACAAGTTCAATGGATGCAGGGATTAC
44
191-211 (F1)

----*---*-*--*-----

128-146 (F2)

LMO84 BIP
AGAAACCATGTTCAAATTGCAAGAGCTTTCTGGACGGCTATC
45
220-243 (B1)

*--------*--------

283-300 (B2)

SEQ ID NO: 58 shows the sequence of the F2 portion in the 3′-side of FIP, and SEQ ID NO: 59 shows the sequence of the B2 portion in the 3′-side of BIP. In the F2 portion and the B2 portion, * represents a base showing polymorphism based on comparison among the sequences of SEQ ID NOs: 1 to 25.

TABLE 11

lmo2736 LAMP primer set 1

SEQ

Sequence (5′ → 3′)
ID NO.
Setting position

LMO2736-1 F3
GAACTAGCCTACATTGATGC
46
508-527

LMO2736-1 B3
TTGAACCGCTTAATAAGTCTG
47
788-808

LMO2736-1 FIP
TTCGTCACGTCACAGGCTATCAGCAACCTTAACCCAAAG
48
568-587 (F1)

------*-----*------

528-546 (F2)

LMO2736-1 BIP
GGAGCAAAACTCGACCAATTTTCGTCCAGGAGCGATACCAC
49
688-710 (B1)

----*-------------

752-769 (B2)

SEQ ID NO: 60 shows the sequence of the F2 portion in the 3′-side of FIP, and SEQ ID NO: 61 shows the sequence of the 82 portion in the 3′-side of BIP. In the F2 portion and the B2 portion, * represents a base showing polymorphism based on comparison among the sequences of SEQ ID NOs: 1 to 25.

TABLE 12

lmo2736 LAMP primer set 2

SEQ

Sequence (5′ → 3′)
ID NO.
Setting position

LMO2736-2 F3
CAAGAACTAGCCTACATTGATG
50
505-526

LMO2736-2 B3
TCTGCATTTAGGAAGGGCATT
51
771-791

LMO2736-2 FIP
TTCGTCACGTCACAGGCTATCAGCAACCTTAACCCAAAGC
52
568-587 (F1)

------*-----*-------

528-547 (F2)

LMO2736-2 BIP
GGAGCAAAACTCGACCAATTTTC-GTCCAGCAGCGATACCAC
53
688-710 (B1)

----*-------------

752-769 (B2)

SEQ ID NO: 62 shows the sequence of the F2 portion in the 3′-side of FIP, and SEQ ID NO: 61 shows the sequence of the B2 portion in the 3′-side of BIP. In the F2 portion and the B2 portion, * represents a base showing polymorphism based on comparison among the sequences of SEQ ID NOs: 1 to 25.

TABLE 13

lmo2736 LAMP primer set 10

SEQ

Sequence (5′ → 3′)
ID NO.
Setting position

LMO2736-10 F3
GTGGCATTCATTTGCAAGAAC
54
491-511

LMO2736-10 B3
GAGCTGAACCGCTTAATAAGTC
55
790-811

LMO2736-10 FIP
GAAGTGGATTCGTCACGTCACAGGCTACATTGATGCCAGCAACCTTAAC
56
572-595 (F1)

*--*--------------*-----*

516-540 (F2)

LMO2736-10 BIP
CTCGACCAATTTTCTTCTCAAAAAATCACCACCAGCGGCTCCG
57
697-724 (B1)

-----*--------*

741-755 (B2)

SEQ ID NO: 63 shows the sequence of the F2 portion in the 3′-side of FIP, and SEQ ID NO: 64 shows the sequence of the B2 portion in the 3′-side of BIP. In the F2 portion and the B2 portion. * represents a base showing polymorphism based on comparison among the sequences of SEQ ID NOs: 1 to 25.

TABLE 14-2

Bacteria belonging to the genus Listeria
LMO2736

other than the monocytogenes bacterium
set
set
set

No.
Bacterial strain
Halo
1
2
10
LMO0084

11

L. ivanovii

GTC02961
(+)
+
+
+
+

12

L. ivanovii
JMC7681
(+)
+
+
+
+

subsp. Ivanovii

13

L. ivanovii

GTC01640T
(+)
+
+
+
+

subsp. Ivanovii

14

L. ivanovii
GTC01641
(+/−)
+
+
+
+

subsp. Iondoniensis

15

L. innocua

GTC16426T
(+)
+
+
+
+

16

L. innocua

GTC02960
(+)
+
+
+
+

17

L. welshimeri

GTC02963T
(+)
+
+
+
+

18

L. seeligeri

GTC16428T
(+)
+
+
+
+

19

L. grayi

GTC02964T
(+)
+
+
+
+

20

L. murrayi

GTC02964
(+)
+
+
+
+

21

L. marthii
GTC16430T

22

L. rocourtiae
GTC16429T

TABLE 14-1

Listeria monocytogenes

LMO2736
LMO

No.
Bacterial strain
Halo
set 1
set 2
set 10
0084

1

L. monocytogenes

1/2a
GTC
(+)
+
+
+
+

02947

2

L. monocytogenes

1/2b
GTC
(+)
+
+
+
+

02948

3

L. monocytogenes

1/2c
JMC
(+)
+
+
+
+

7672

4

L. monocytogenes

3a
JMC
(+/−)
+
+
+
+

7673

5

L. monocytogenes

3b
JMC
(+)
+
+
+
+

7677

6

L. monocytogenes

3c
JMC
(+)
+
+
+
+

7678

7

L. monocytogenes

4a
JMC
(+)
+
+
+
+

7674

8

L. monocytogenes

4b
JMC
(+)
+
+
+
+

7675

9

L. monocytogenes

4d
JMC
(+)
+
+
+
+

7680

10

L. monocytogenes

5
GTC
(+)
+
+
+
+

02957

TABLE 14-3

Food-poisoning bacteria other than
LMO2736

bacteria belonging to the genus Listeria
set
set
set

No.
Bacterial strain
1
2
10
LMO0084

23

Escherichia coli

ATCC10798
−
−
−
−

24

Salmonella subsp.
JA.107
−
−
−
−

enterica (I)

25

Salmonella subsp.
JA.125
−
−
−
−

salamae (II)

26

Salmonella subsp.
JA.76
−
−
−
−

arizonae (IIIa)

27

Salmonella subsp.
JA.129
−
−
−
−

diarizinae (IIIb)

28

Salmonella subsp.
JA.n-22
−
−
−
−

houtenae (IV)

29

Salmonella bongori (V)
JA.94
−
−
−
−

30

Salmonella subsp.
ATCC43971
−
−
−
−

enterica Typhimurium

31

Staphylococcus aureus

ATCC6538P
−
−
−
−

32

Staphylococcus aureus

ATCC25923
−
−
−
−

33

Staphylococcus aureus

ATCC29213
−
−
−
−

34

Staphylococcus aureus

JMC2197
−
−
−
−

35

Staphylococcus aureus

IMCB.IMA2
−
−
−
−

36

Staphylococcus cohnii

ATCC29974
−
−
−
−

37

Staphylococcus

ATCC29970
−
−
−
−

haemolyticus

38

Staphylococcus hyicus

ATCC11249
−
−
−
−

subsp.

39

Staphylococcus

ATCC29663
−
−
−
−

intermedius

40

Staphylococcus

ATCC15305
−
−
−
−

saprophyticus

41

Citrobacter freundii

ATCC8090
−
−
−
−

42

Citrobacter freundii

ATCC8043
−
−
−
−

43

Proteus vulgaris

IFO3988
−
−
−
−

44

Lactobacillus

IFO13953
−
−
−
−

bulgaricus

45

Lactobacillus

IFO3809
−
−
−
−

helveticus

46

Streptococcus sp.
IFO3535
−
−
−
−

47

Streptococcus

ATCC10558
−
−
−
−

sanguis

48

Streptococcus mitis

ATCC6249
−
−
−
−

For covering polymorphic sequences of more serotypes, mixed primers using mixed bases were designed at the LMO0084 primer designing sites shown above in Table 6.

TABLE 15

SEQ

LMO0084
ID

primer
NO.
Sequence
Serotype

F286A
26
AGCCGTCCAGAAAGCATCAA
1/2a, 1/2b, 1/2c,

3a, 3b, 3c, 4b, 4e

F286B
27
AGCCGCCCAGAAAGTCTCAA
4a, 4c

F286/M
67
AGCCGYCCAGAAAGYMTCAA

F281A
28
TCGATAGCCGTCCAGAAAGC
1/2a, 1/2b, 1/2c,

3a, 3b, 3c, 4b, 4e

F281B
29
TTGATAGCCGCCCAGAAAGT
4a, 4e

F281/M
68
TYGATAGCCGYCCAGAAAGY

R757A
30
GCTCGTCGGCGATTTCTTTC
1/2a, 1/2c, 3a, 3c

R757B
31
GCTCGTCGGCTATTTCTTTC
1/2b, 3b, 3c, 4a,

4b, 4c

R757
See
GCTCGTCAGCTATTTCTTTC
4c

9

R757/M
69
GCTCGTCRGCKATTTCTTTC

Ordinary PCR was carried out with the combinations of F286/M and R757/M, and 281/M and R757/M, to see whether monocytogenes-specific amplification can be found therewith. Detection tests were carried out using the monocytogenes bacterial strains of the bacterial strain Nos. 1 to 10 shown in Table 5-1, the bacterial strains of the bacterial strain Nos. 11 to 22 belonging to the genus Listeria shown in Table 5-2, and the food-poisoning bacteria of the bacterial strain Nos. 23 to 48 (wherein, however, the Citrobacter freundii N-326 strain was used instead of the bacterial strain No. 42).

As a result, all monocytogenes bacteria showed amplification, and none of the bacterial strains other than the monocytogenes bacteria showed amplification (Tables 16-1 to 16-6).

TABLE 16-1

1
2
3
4
5
6

monocytogenes

monocytogenes

monocytogenes

monocytogenes

monocytogenes

monocytogenes

LM00084
1/2a
1/2b
1/2c
3a
3b
3c

No.
primer: F
primer: R
size
GTC02947
GTC02948
JMC7672
JMC7673
JMC7677
JMC7678

1
F286/M
R757/M
471
+
+
+
+
+
+

2
F281/M
R757/M
476
+
+
+
+
+
+

7
8
9
10

monocytogenes

monocytogenes

monocytogenes

monocytogenes

LM00084
4a
4b
4d
5

No.
primer: F
primer: R
size
JMC7674
JMC7675
JMC7680
GTC02957

1
F286/M
R757/M
471
+
+
+
+

2
F281/M
R757/M
476
+
+
+
+

TABLE 16-2

12
13
14

ivanovii

ivanovii

ivanovii

11
subsp.
subsp.
subsp.
15
16

LMO0084

ivanovii

Ivanovii

Ivanovii

londoniensisi

innocua

innocua

No.
primer: F
primer: R
size
GTC02961
JMC7681
GTC01640T
GTC01641
GTC16426T
GTC02960

1
F286/M
R757/M
471
−
−
−
−
−
−

2
F281/M
R757/M
476
−
−
−
−
−
−

17
18
19
20

LMO0084

welshimeri

seeligeri

grayi

murrayi

No.
primer: F
primer: R
size
GTC02963T
GTC16428T
GTC02964T
GTC02964

1
F286/M
R757/M
471
−
−
−
−

2
F281/M
R757/M
476
−
−
−
−

TABLE 16-3

21
22

LMO0084

marthii

rocourtiae

No.
primer: F
primer: R
size
GTC16430T
GTC16429T

1
F286/M
R757/M
471
−
−

2
F281/M
R757/M
476
−
−

TABLE 16-4

24
25
26
27
28

23

Salmonella

Salmonella

Salmonella

Salmonella

Salmonella

Escherichia

subsp.
subsp.
subsp.
subsp.
subsp.

coli

Enterica

Salamae

Arizonae

Diarizinae

Houtenae

LMO0084
(K12)
(I)
(II)
(IIIa)
(IIIb)
(IV)

No.
primer: F
primer: R
size
ATCC10798
JA.107
JA.125
JA.76
JA.129
JA.n-22

1
F286/M
R757/M
471
−
−
−
−
−
−

2
F281/M
R757/M
476
−
−
−
−
−
−

30

29

Salmonella

Salmonella

subsp.
31
32

bongori

Enterica

Staphylococcus

Staphylococcus

LMO0084
(V)

Typhimurium

aureus

aureus

No.
primer: F
primer: R
size
JA.94
ATCC43971
ATCC6538P
ATCC25923

1
F286/M
R757/M
471
−
−
−
−

2
F281/M
R757/M
476
−
−
−
−

TABLE 16-5

33
34
35
36
40
37

Staphylococcus

Staphylococcus

Staphylococcus

Staphylococcus

Staphylococcus

Staphylococcus

LMO0084

aureus

aureus

aureus

cohnii

saprophyticus

haemolyticus

No.
primer: F
primer: R
size
ATCC29213
JMC2197
IMCB.IMA2
ATCC29974
ATCC15305
ATCC29970

1
F286/M
R757/M
471
−
−
−
−
−
−

2
F281/M
R757/M
476
−
−
−
−
−
−

38
39
41

Staphylococcus

Staphylococcus

Citrobacter

Citrobacter

LMO0084

hyicus subsp.

intermedius

freundii

freundii

No.
primer: F
primer: R
size
ATCC11249
ATCC29S63
ATCC8090
N-326

1
F286/M
R757/M
471
−
−
−
−

2
F281/M
R757/M
476
−
−
−
−

TABLE 16-6

43
44
45
46
47
48

Proteus

Lactbacillus

Lactbacillus

Streptcoccus

Streptcoccus

Streptcoccus

LMO0084

vulgaris

bulgarius

helvelicus

sp.

sanguis

mitis

No.
primer: F
primer: R
size
IFO3988
IFO13953
IFO3809
IFO3535
ATCC10558
ATCC6249

1
F286/M
R757/M
471
−
−
−
−
−
−

2
F281/M
R757/M
476
−
−
−
−
−
−

Similarly, for LMO2736, mixed primers using mixed bases were designed at the lmo2736 primer designing sites shown above in Table 6. The “SEQ ID NO.” column shows the SEQ ID NOs. of the lmo2736 gene of the referred serotypes and the SEQ ID NOs. describing the primer sequences.

TABLE 17

Position

Primer

abbreviations

SEQ ID

F/R
are shown in ()
Sequence
NO.
Serotype

F
8-27
TCGTCATCGCACCTGATTCA
32
1/2a, 1/2c, 3a, 3b, 3c, 4a, 4b,

(F8)

4c, 4d, 4e

F
222-241
GGCCTCCTACGGTATTCACG
15 etc
1/2c, 3a, 3c

F
222-241
GGCCCCCTACGGTATTCACA
13
1/2a

F
222-241
GGCCTCCTACGGTATTCACA
19, 22
4a, 4c

F
222-241
CCCCTCCTACGGTATTCTCG
14 etc
1/2b, 3b, 4b, 4d, 4e

F
222Mix
GGCCYCCTACGGTATTCWCR
70

(F222/M)

F
488-507
CCGGTGGCATTCATTTGCAA
14 etc
1/2b, 1/2c, 3b, 3c, 4a, 4b, 4c, 4d, 4e

F
488-507
CCGGCGGTATTCATTTCCAA
13, 16
1/2a, 3a

F
488Mix
CCGGYGGYATTCATTTGCAA
71

(F488/M)

F
530-549
GCAACCTTAACCCAAAGCTG
15, 18
1/2c, 3c

F
530-549
GCAATCTTAACCCAAAGCTG
13, 16
1/2a, 3a

F
530-549
GCAACCTTAATCCAAACCTG
14 etc
1/2b, 3b, 4a, 4b, 4c, 4d, 4e

F
530Mix
GCAAYCTTAAYCCAAAGCTG
72

(F530/M)

F
572-591
CCTGTGACGTGACGAATCCA
13 etc
1/2a, 1/2c, 3a, 3c

F
572-591
CCTGCGACGTCACGAATCCA
14 etc
1/2b, 3b, 4b, 4c, 4d, 4e

F
572-591
CCTGTGACGTCACGAATCCA
19
4a

F
572Mix
CCTGYGACGTSACGAATCCA
73

(F572/M)

R
176-157
TCCACCTCGGAAGACTCACT
37
1/2a, 1/2b, 1/2c, 3a, 3b, 3c, 4a, 4b,

(R176)

4c, 4d, 4e

R
591-572
TGGATTCGTCACGTCACAGG
13 etc
1/2a, 1/2c, 3a, 3c

R
591-572
TGGATTCGTGACGTCGCAGG
14 etc
1/2b, 3b, 4b, 4c, 4d, 4e

R
591-572
TCCATTCGTGACGTCACAGG
19
4a

R
591Mix
TGGATTCGTSACGTCRCAGG
74

(R591/M)

R
685-666
AGTTCTGCATGGCGTTCTCT
13 etc
1/2a, 1/2c, 3a, 3c

R
685-666
AGTTCTGCATCCCCCGCTCT
14 etc
1/2b, 3b, 4b, 4d, 43

R
685-666
AGTTCTGCATGGCGTGCTCT
19
4a

R
685-666
AGTTCTGCATGGCATGCTCT
22
4c

R
685Mix
AGTTCTGCATGGCRYKCTCT
75

(R685/M)

R
771-752
TAGTCCAGCAGCGATACCAC
13 etc
1/2a, 1/2c, 3a, 3c

R
771-752
TAGTCCGGCAGCGATACCAC
14 etc
1/2b, 3b, 4a, 4b, 4c, 4d, 4e

R
771Mix
TAGTCCRGCAGCGATACCAC
76

(R771 /M)

R
992-973
TTGTTTTCGAGTGCAAGGCT
41
1/2a, 1/2b, 1/2c, 3a, 3b, 3c, 4a, 4b,

(R992)

4c, 4d, 4e

Ordinary PCR was carried out with the combinations of an F primer and an R primer shown below in Table 18 to see whether monocytogenes-specific amplification can be found therewith. Detection tests were carried out using the monocytogenes bacterial strains of the bacterial strain Nos. 1 to 10 shown in Table 5-1, the bacterial strains of the bacterial strain Nos. 11 to 22 belonging to the genus Listeria shown in Table 5-2, and the food-poisoning bacteria of the bacterial strain Nos. 23 to 48 (wherein, however, the Citrobacter freundii N-326 strain was used instead of the bacterial strain No. 42).

As a result, all monocytogenes bacteria showed amplification, and none of the bacterial strains other than the monocytogenes bacteria showed amplification (Tables 19-1 to 19-6).

TABLE 18

LMO2736
LMO2736
Amplification

F primer
R primer
size

1
F8
R176
168

2
F222/M
R591/M
369

3
F488/M
R591/M
103

4
F488/M
R685/M
197

5
F488/M
R771/M
283

6
F488/M
R992
504

7
F530/M
R685/M
155

8
F530/M
R771/M
241

9
F530/M
R992
462

10
F572/M
R685/M
133

11
F572/M
R771/M
199

12
F572/M
R992
420

TABLE 19-1

1
2
3
4
5
6

monocytogenes

monocytogenes

monocytogenes

monocytogenes

monocytogenes

monocytogenes

LMO2736
1/2a
1/2b
1/2c
3a
3b
3c

No.
primer: F
primer: R
size
GTC02947
GTC02948
JMC7672
JMC7673
JMC7677
JMC7678

1
F8
R176
168
+
+
+
+
+
+

2
F222/M
R591/M
369
+
+
+
+
+
+

3
F488/M
R591/M
103
+
+
+
+
+
+

4
F488/M
R685/M
197
+
+
+
+
+
+

5
F488/M
R771/M
283
+
+
+
+
+
+

6
F488/M
R992
504
+
+
+
+
+
+

7
F530/M
R685/M
155
+
+
+
+
+
+

8
F530/M
R771/M
241
+
+
+
+
+
+

9
F530/M
R992
462
+
+
+
+
+
+

10
F572/M
R685/M
133
+
+
+
+
+
+

11
F572/M
R771/M
199
+
+
+
+
+
+

12
F572/M
R992
420
+
+
+
+
+
+

7
8
9
10

monocytogenes

monocytogenes

monocytogenes

monocytogenes

LMO2736
4a
4b
4d
5

No.
primer: F
primer: R
size
JMC7674
JMC7675
JMC7680
GTC02957

1
F8
R176
168
+
+
+
+

2
F222/M
R591/M
369
+
+
+
+

3
F488/M
R591/M
103
+
+
+
+

4
F488/M
R685/M
197
+
+
+
+

5
F488/M
R771/M
283
+
+
+
+

6
F488/M
R992
504
+
+
+
+

7
F530/M
R685/M
155
+
+
+
+

8
F530/M
R771/M
241
+
+
+
+

9
F530/M
R992
462
+
+
+
+

10
F572/M
R685/M
133
+
+
+
+

11
F572/M
R771/M
199
+
+
+
+

12
F572/M
R992
420
+
+
+
+

TABLE 19-2

12
13
14

ivanovii

ivanovii

ivanovii

11
subsp.
subsp.
subsp.
15
16

LMO2736

ivanovii

Ivanovii

Ivanovii

londoniensisi

innocua

innocua

No.
primer: F
primer: R
size
GTC02961
JMC7681
GTC01640T
GTC01641
GTC16426T
GTC02960

1
F8
R176
168
−
−
−
−
−
−

2
F222/M
R591/M
369
−
−
−
−
−
−

3
F488/M
R591/M
103
−
−
−
−
−
−

4
F488/M
R685/M
197
−
−
−
−
−
−

5
F488/M
R771/M
283
−
−
−
−
−
−

6
F488/M
R992
504
−
−
−
−
−
−

7
F530/M
R685/M
155
−
−
−
−
−
−

8
F530/M
R771/M
241
−
−
−
−
−
−

9
F530/M
R992
462
−
−
−
−
−
−

10
F572/M
R685/M
133
−
−
−
−
−
−

11
F572/M
R77/M
199
−
−
−
−
−
−

12
F572/M
R992
420
−
−
−
−
−
−

17
18
19
20

LMO2736

welshimeri

seeligeri

grayi

murrayi

No.
primer: F
primer: R
size
GTC02963T
GTC16428T
GTC02964T
GTC02964

1
F8
R176
168
−
−
−
−

2
F222/M
R591/M
369
−
−
−
−

3
F488/M
R591/M
103
−
−
−
−

4
F488/M
R685/M
197
−
−
−
−

5
F488/M
R771/M
283
−
−
−
−

6
F488/M
R992
504
−
−
−
−

7
F530/M
R685/M
155
−
−
−
−

8
F530/M
R771/M
241
−
−
−
−

9
F530/M
R992
462
−
−
−
−

10
F572/M
R685/M
133
−
−
−
−

11
F572/M
R77/M
199
−
−
−
−

12
F572/M
R992
420
−
−
−
−

TABLE 19-3

21
22

LMO2736

marthii

rocourtiae

No.
primer: F
primer: R
size
GTC16430T
GTC16429T

1
F8
R176
168
−
−

2
F222/M
R591/M
369
−
−

3
F488/M
R591/M
103
−
−

4
F488/M
R685/M
197
−
−

5
F488/M
R771/M
283
−
−

6
F488/M
R992
504
−
−

7
F530/M
R685/M
155
−
−

8
F530/M
R771/M
241
−
−

9
F530/M
R992
462
−
−

10
F572/M
R685/M
133
−
−

11
F572/M
R771/M
199
−
−

12
F572/M
R992
420
−
−

TABLE 19-4

24
25
26
27
28

23

Salmonella

Salmonella

Salmonella

Salmonella

Salmonella

Escherichia

subsp.
subsp.
subsp.
subsp.
subsp.

coli

Enterica

Salamae

Arizonae

Diarizinae

Houtenae

LMO2736
(K12)
(I)
(II)
(IIIa)
(IIIb)
(IV)

No.
primer: F
primer: R
size
ATCC10798
JA.107
JA.125
JA.76
JA.129
JA.n-22

1
F8
R176
168
−
−
−
−
−
−

2
F222/M
R591/M
369
−
−
−
−
−
−

3
F488/M
R591/M
103
−
−
−
−
−
−

4
F488/M
R685/M
197
−
−
−
−
−
−

5
F488/M
R771/M
283
−
−
−
−
−
−

6
F488/M
R992
504
−
−
−
−
−
−

7
F530/M
R685/M
155
−
−
−
−
−
−

8
F530/M
R771/M
241
−
−
−
−
−
−

9
F530/M
R992
462
−
−
−
−
−
−

10
F572/M
R685/M
133
−
−
−
−
−
−

11
F572/M
R771/M
199
−
−
−
−
−
−

12
F572/M
R992
420
−
−
−
−
−
−

30

29

Salmonella

Salmonella

subsp.
31
32

bongori

Enterica

Staphylococcus

Staphylococcus

LMO2736
(V)

Typhimurium

aureus

aureus

No.
primer: F
primer: R
size
JA.94
ATCC43971
ATCC6538P
ATCC25923

1
F8
R176
168
−
−
−
−

2
F222/M
R591/M
369
−
−
−
−

3
F488/M
R591/M
103
−
−
−
−

4
F488/M
R685/M
197
−
−
−
−

5
F488/M
R771/M
283
−
−
−
−

6
F488/M
R992
504
−
−
−
−

7
F530/M
R685/M
155
−
−
−
−

8
F530/M
R771/M
241
−
−
−
−

9
F530/M
R992
462
−
−
−
−

10
F572/M
R685/M
133
−
−
−
−

11
F572/M
R771/M
199
−
−
−
−

12
F572/M
R992
420
−
−
−
−

TABLE 19-5

33
34
35
36
40
37

Staphylococcus

Staphylococcus

Staphylococcus

Staphylococcus

Staphylococcus

Staphylococcus

LMO2736

aureus

aureus

aureus

cohnii

saprophyticus

haemolyticus

No.
primer: F
primer: R
size
ATCC29213
JMC2197
IMCB.IMA2
ATCC29974
ATCC15305
ATCC29970

1
F8
R176
168
−
−
−
−
−
−

2
F222/M
R591/M
369
−
−
−
−
−
−

3
F488/M
R591/M
103
−
−
−
−
−
−

4
F488/M
R685/M
197
−
−
−
−
−
−

5
F488/M
R771/M
283
−
−
−
−
−
−

6
F488/M
R992
504
−
−
−
−
−
−

7
F530/M
R685/M
155
−
−
−
−
−
−

8
F530/M
R771/M
241
−
−
−
−
−
−

9
F530/M
R992
462
−
−
−
−
−
−

10
F572/M
R685/M
133
−
−
−
−
−
−

11
F572/M
R771/M
199
−
−
−
−
−
−

12
F572/M
R992
420
−
−
−
−
−
−

38
39
41

Staphylococcus

Staphylococcus

Citrobacter

Citrobacter

LMO2736

hyicus subsp.

intermedius

freundii

freundii

No.
primer: F
primer: R
size
ATCC11249
ATCC29663
ATCC8090
N-326

1
F8
R176
168
−
−
−
−

2
F222/M
R591/M
369
−
−
−
−

3
F488/M
R591/M
103
−
−
−
−

4
F488/M
R685/M
197
−
−
−
−

5
F488/M
R771/M
283
−
−
−
−

6
F488/M
R992
504
−
−
−
−

7
F530/M
R685/M
155
−
−
−
−

8
F530/M
R771/M
241
−
−
−
−

9
F530/M
R992
462
−
−
−
−

10
F572/M
R685/M
133
−
−
−
−

11
F572/M
R771/M
199
−
−
−
−

12
F572/M
R992
420
−
−
−
−

TABLE 19-6

43
44
45
46
47
48

Proteus

Lactbacillus

Lactbadllus

Streptcoccus

Streptcoccus

Streptooccus

LMO2736

vulgaris

bulgarius

helveticus

sp.

sanguis

mitis

No.
primer: F
primer: R
size
IFO3988
IFO13953
IFO3809
IFO3535
ATCC10558
ATCC6249

1
F8
R176
168
−
−
−
−
−
−

2
F222/M
R591/M
369
−
−
−
−
−
−

3
F488/M
R591/M
103
−
−
−
−
−
−

4
F488/M
R685/M
197
−
−
−
−
−
−

5
F488/M
R771/M
283
−
−
−
−
−
−

6
F488/M
R992
504
−
−
−
−
−
−

7
F530/M
R685/M
155
−
−
−
−
−
−

8
F530/M
R771/M
241
−
−
−
−
−
−

9
F530/M
R992
462
−
−
−
−
−
−

10
F572/M
R685/M
133
−
−
−
−
−
−

11
F572/M
R771/M
199
−
−
−
−
−
−

12
F572/M
R992
420
−
−
−
−
−
−

Aiming at construction of a real-time PCR detection system, TaqMan (registered trademark) probes were designed.

[1] LMO0084 Gene

A TaqMan (registered trademark) probe is commonly designed under the following conditions.

- A TaqMan (registered trademark) probe is designed as a 20-mer to 30-mer probe (citation from Thermo Fisher).
- The amplification target ideally has a length of 70 bp to 200 bp, and should have a length of less than 300 bp (citation from QIAGEN).

However, according to the sets of mixed primers targeting the LMO0084 gene, designed as described above (LMO0084-F286/M and LMO0084-R757/M, and LMO0084-F281/M and LMO0084-R757/M), the length of the amplification target was about 470 bp. Since specificity in the PCR could not be obtained with a length shorter than this, TaqMan (registered trademark) probes were designed within this range. In the amplification target, 20-mer or longer sequences containing a common sequence of not more than two bases in the monocytogenes bacterium were present at three locations (Table 20).

TABLE 20

Number

Probe
of bases
Characteristics

TMP366-389
23 mer
Common to all sequences

TMP535-558
23 mer
TA-type and CC-type exist

TMP686-711
26 mer
Common to all sequences

In view of this, the following four kinds of sequences were employed as probe sequences. The oligonucleotide having each sequence was modified with the fluorescent substance FAM (6-carboxyfluorescein) at the 5′-end, and with the quencher substance TAMRA at the 3′-end, to prepare a TaqMan (registered trademark) probe.

(SEQ ID NO: 77)

0084TMP366-389:
TATTACATTCATAGAATTGACCC

(SEQ ID NO: 78)

0084TMP535-558(TA):
ATCTGGTGGCGAGAAGCTGAAAA

(SEQ ID NO: 79)

0084TMP535-558(CC):
ATCTGGTGGCGAGAAGCCGAACA

(SEQ ID NO: 80)

0084TMP686-711:
TACCAAGATTCCAAAAAGAAGCCATG

The sets of mixed primers shown in Table 15 (LMO0084-F286M and LMO0084-R757M, and LMO0084-F281M and LMO0084-R757M) were used in combination with these TaqMan (registered trademark) probes to carry out detection experiments by real-time PCR using the genomes of test bacterial strains as templates. As the test bacterial strains, the monocytogenes bacterial strains of the bacterial strain Nos. 1 to 10 shown in Table 5-1, the bacterial strains of the bacterial strain Nos. 11 to 22 belonging to the genus Listeria shown in Table 5-2, and the food-poisoning bacteria of the bacterial strain Nos. 23 to 48 (wherein, however, the Citrobacter freundii N-326 strain was used instead of the bacterial strain No. 42) were used.

TABLE 21

[Composition of the reaction liquid for

real-time PCR (total 20 μL)]

Template DNA (1 ng/μL)
1.00 μL

TaqMan Fast Advanced Master Mix(2x)
10.00 μL

100 μM Primer F
0.08 μL (per primer

sequence)

100 μM Primer R
0.08 μL (per primer

sequence)

100 μM TaqMan probe
0.25 μL

Distilled Water
Appropriate volume

[Reaction Conditions]

Apparatus used: Corbett Research: Roter-Gene6000

50° C. for 2 minutes (holding)→95° C. for 2 minutes (holding)→(95° C. for 3 seconds −64° C. for 15 seconds)×40 cycles

Evaluation was carried out based on the presence or absence of the amplification curve. Agarose electrophoresis of the PCR product was also carried out, and the presence or absence of a band, and the band size were investigated.

The results of the real-time PCR tests are shown in Tables 22-1 to 22-3. 0084TMP366-389 and 0084TMP686-711 were capable of specific detection of the monocytogenes bacterium by combination with either primer set. 0084TMP535-558(TA) and 0084TMP535-558(CC) were found to be similarly capable of specific detection of the monocytogenes bacterium when they were used as a mixed probe. In cases where these are used as a mixture, the reaction liquid composition may be 0.25 μL of 100 μM 0084TMP535-558(TA) and 0.25 μL of 100 μM 0084TMP535-558(CC).

TABLE 22-1

TaqMan ® probe

TMP366-389
TMP535-558
TMP535-558
TMP535-558
TMP686-711
TMP366-389

−
TA
CC
Mix (TA/CC)
−
−

primer F

F286/M
F286/M
F286/M
F286/M
F286/M
F281/M

primer: R

R757/M
R757/M
R757/M
R757/M
R757/M
R757/M

LMO 0084
size
471
476
476
476
471
476

1

Listeria

1/2a
GTC02947
+
+
−
+
+
+

monocytogenes

2

Listeria

1/2b
GTC02948
+
+
−
+
+
+

monocytogenes

3

Listeria

1/2c
JMC7672
+
+
−
+
+
+

monocytogenes

4

Listeria

3a
JMC7673
+
+
−
+
+
+

monocytogenes

5

Listeria

3b
JMC7677
+
+
−
+
+
+

monocytogenes

6

Listeria

3c
JMC7678
+
+
−
+
+
+

monocytogenes

7

Listeria

4a
JMC7674
+
−
+
+
+
+

monocytogenes

8

Listeria

4b
JMC7675
+
+
−
+
+
+

monocytogenes

9

Listeria

4d
JMC7680
+
+
−
+
+
+

monocytogenes

10

Listeria

5
GTC02357
+
+
−
+
+
+

monocytogenes

TaqMan ® probe

TMP535-558
TMP535-558
TMP535-558
TMP686-711

TA
CC
Mix (TA/CC)
−

primer F

F281/M
F281/M
F281/M
F281/M

primer H

R757/M
R757/M
R757/M
R757/M

LMO 0084
size
476
476
476
476

1

Listeria

1/2a
GTC02947
+
−
+
+

monocytogenes

2

Listeria

1/2b
GTC02948
+
−
+
+

monocytogenes

3

Listeria

1/2c
JMC7672
+
−
+
+

monocytogenes

4

Listeria

3a
JMC7673
+
−
+
+

monocytogenes

5

Listeria

3b
JMC7677
+
−
+
+

monocytogenes

6

Listeria

3c
JMC7678
+
−
+
+

monocytogenes

7

Listeria

4a
JMC7674
−
+
+
+

monocytogenes

8

Listeria

4b
JMC7675
+
−
+
+

monocytogenes

9

Listeria

4d
JMC7680
+
−
+
+

monocytogenes

10

Listeria

5
GTC02357
+
−
+
+

monocytogenes

TABLE 22-2

TaqMan ® probe

TMP366-389
TMP535-558
TMP535-558
TMP535-558
TMP686-711
TMP366-389

−
TA
CC
Mix (TA/CC)
−
−

primer F

F286/M
F286/M
F286/M
F286/M
F286/M
F281/M

primer R

R757/M
R757/M
R757/M
R757/M
R757/M
R757/M

LMO 0084
size
471
471
471
471
471
476

11

Listeria ivanovii

GTC02961
−
−
−
−
−
−

12

Listeria ivanovii

JMC7681
−
−
−
−
−
−

subsp. Ivanovii

13

Listeria ivanovii

GTC01640T
−
−
−
−
−
−

subsp. Ivanovii

14

Listeria ivanovii

GTC01641
−
−
−
−
−
−

subsp. londonionsisi

15

Listeria innocua

GTC16426T
−
−
−
−
−
−

16

Listeria innocua

GTC02960
−
−
−
−
−
−

17

Listeria welshimeri

GTC02963T
−
−
−
−
−
−

18

Listeria seeligeri

GTC16428T
−
−
−
−
−
−

19

Listeria grayi

GTC02964T
−
−
−
−
−
−

20

Listeria murrayi

GTC02964
−
−
−
−
−
−

21

Listeria marthii

GTC16430T
−
−
−
−
−
−

22

Listeria rocourtiae

GTC16429T
−
−
−
−
−
−

TaqMan ® probe

TMP535-558
TMP535-558
TMP686-711

TMP535-558
CC
Mix (TA/CC)
−

TA
primer F

F281/M
F281/M
F281/M
F281/M

primer R

R757/M
R757/M
R757/M
R757/M

LMO 0084
size
476
476
476
476

11

Listeria ivanovii

GTC02961
−
−
−
−

12

Listeria ivanovii

JMC7681
−
−
−
−

subsp. Ivanovii

13

Listeria ivanovii

GTC01640T
−
−
−
−

subsp. Ivanovii

14

Listeria ivanovii

GTC01641
−
−
−
−

subsp. londonionsisi

15

Listeria innocua

GTC16426T
−
−
−
−

16

Listeria innocua

GTC02960
−
−
−
−

17

Listeria welshimeri

GTC02963T
−
−
−
−

18

Listeria seeligeri

GTC16428T
−
−
−
−

19

Listeria grayi

GTC02964T
−
−
−
−

20

Listeria murrayi

GTC02964
−
−
−
−

21

Listeria marthii

GTC16430T
−
−
−
−

22

Listeria rocourtiae

GTC16429T
−
−
−
−

TABLE 22-3

TaqMan ® probe

TMP366-389
TMP535-558
TMP535-558
TMP535-558
TMP686-711
TMP366-389

−
TA
CC
Mix (TA/CC)
−
−

primer F

F286/M
F286/M
F28/M
F286/M
F286/M
F281/M

primer R

R757/M
R757/M
R757/M
R757/M
R757/M
R757/M

LMO 0084
size
471
471
471
471
471
476

23

Escherichia coli (K12)
ATCC10798
−
−
−
−
−
−

24

Salmonella subsp.
JA.107
−
−
−
−
−
−

Enterica (I)

25

Salmonella subsp.
JA.125
−
−
−
−
−
−

Salamae (II)

26

Salmonella subsp.
JA.76
−
−
−
−
−
−

Arizonae (IIIa)

27

Salmonella subsp.
JA.129
−
−
−
−
−
−

Diarizinae (IIIb)

28

Salmonella subsp.
JA.n-22
−
−
−
−
−
−

Houtenae (IV)

29

Salmonella bongori (V)
JA.94
−
−
−
−
−
−

30

Salmonella subsp.
ATCC43971
−
−
−
−
−
−

EntericaTyphimurium

31

Staphylococcus

ATCC6538P
−
−
−
−
−
−

aureus

32

Staphylococcus

ATCC25923
−
−
−
−
−
−

aureus

33

Staphylococcus

ATCC29213
−
−
−
−
−
−

aureus

34

Staphylococcus

JMC2197
−
−
−
−
−
−

aureus

35

Staphylococcus

IMCB.IMA2
−
−
−
−
−
−

aureus

36

Staphylococcus

ATCC29974
−
−
−
−
−
−

cohnii

40

Staphylococcus

ATCC15305
−
−
−
−
−
−

saprophyticus

37

Staphylococcus

ATCC29970
−
−
−
−
−
−

haemolyticus

38

Staphylococcus

ATCC11249
−
−
−
−
−
−

hyicus subsp.

39

Staphylococcus

ATCC29663
−
−
−
−
−
−

intermedius

41

Citrobacter

ATCC8090
−
−
−
−
−
−

freundii

Citrobacter

N-326
−
−
−
−
−
−

freundii

43

Proteus vulgaris

IFO3988
−
−
−
−
−
−

44

Lactbacillus

IFO13953
−
−
−
−
−
−

bulgarius

45

Lactbacillus

IFO3809
−
−
−
−
−
−

helveticus

46

Streptcoccus sp.
IFO3535
−
−
−
−
−
−

47

Streptcoccus

ATCC10558
−
−
−
−
−
−

sanguis

48

Streptcoccus mitis

ATCC6249
−
−
−
−
−
−

TaqMan ® probe

TMP535-558
TMP535-558
TMP535-558
TMP686-711

TA
CC
Mix (TA/CC)
−

primer F

F281/M
F281/M
F281/M
F281/M

primer R

R757/M
R757/M
R757/M
R757/M

LMO 0084
size
476
476
476
476

23

Escherichia coli (K12)
ATCC10798
−
−
−
−

24

Salmonella subsp.
JA.107
−
−
−
−

Enterica (I)

25

Salmonella subsp.
JA.125
−
−
−
−

Salamae (II)

26

Salmonella subsp.
JA.76
−
−
−
−

Arizonae (IIIa)

27

Salmonella subsp.
JA.129
−
−
−
−

Diarizinae (IIIb)

28

Salmonella subsp.
JA.n-22
−
−
−
−

Houtenae (IV)

29

Salmonella bongori (V)
JA.94
−
−
−
−

30

Salmonella subsp.
ATCC43971
−
−
−
−

EntericaTyphimurium

31

Staphylococcus

ATCC6538P
−
−
−
−

aureus

32

Staphylococcus

ATCC25923
−
−
−
−

aureus

33

Staphylococcus

ATCC29213
−
−
−
−

aureus

34

Staphylococcus

JMC2197
−
−
−
−

aureus

35

Staphylococcus

IMCB.IMA2
−
−
−
−

aureus

36

Staphylococcus

ATCC29974
−
−
−
−

cohnii

40

Staphylococcus

ATCC15305
−
−
−
−

saprophyticus

37

Staphylococcus

ATCC29970
−
−
−
−

haemolyticus

38

Staphylococcus

ATCC11249
−
−
−
−

hyicus subsp.

39

Staphylococcus

ATCC29663
−
−
−
−

intermedius

41

Citrobacter

ATCC8090
−
−
−
−

freundii

Citrobacter

N-326
−
−
−
−

freundii

43

Proteus vulgaris

IFO3988
−
−
−
−

44

Lactbacillus

IFO13953
−
−
−
−

bulgarius

45

Lactbacillus

IFO3809
−
−
−
−

helveticus

46

Streptcoccus sp.
IFO3535
−
−
−
−

47

Streptcoccus

ATCC10558
−
−
−
−

sanguis

48

Streptcoccus mitis

ATCC6249
−
−
−
−

Based on the above results, the following are primer-probe combinations that can be preferably used for detection of the LMO0084 gene by real-time PCR. The number in [ ] represents a SEQ ID NO. in SEQUENCE LISTING.

TABLE 23

LMO0084 primers
LMO0084

primer: F
primer: R
TaqMan (registered trademark) probe

1
F286/M [67]
R757/M [69]
0084TMP366-389 [77]

2
F286/M [67]
R757/M [69]
0084TMP535-558(TA) [78]

0084TMP535-558(CC) [79]

3
F286/M [67]
R757/M [69]
0084TMP686-711 [80]

4
F281/M [68]
R757/M [69]
0084TMP366-389 [77]

5
F281/M [68]
R757/M [69]
0084TMP535-558(TA) [78]

0084TMP535-558(CC) [79]

6
F281/M [68]
R757/M [69]
0084TMP686-711 [80]

[2] LMO2736 Gene

For each of No. 1 and No. 2 among the primer sets shown in Table 18, one TaqMan (registered trademark) probe was designed in the PCR amplification region (Table 24).

Since common sequences were hardly present in the PCR amplification of No. 3 to No. 12, a TaqMan (registered trademark) probe was set at one location in a common sequence in the PCR amplification regions of No. 4 to No. 12 (position 488 to position 992) (Table 24).

TABLE 24

Number

LMO0084 probe
of bases
Characteristics

TMP70-89
20 mer
Common to all sequences

TMP372-393
20 mer
Common to all sequences

TMP619-647
29 mer
GG-type and CC-type exist

The following four kinds of sequences were employed as probe sequences. The oligonucleotide having each sequence was modified with the fluorescent substance FAM (6-carboxyfluorescein) at the 5′-end, and with the quencher substance TAMRA at the 3′-end, to prepare a TaqMan (registered trademark) probe.

(SEQ ID NO: 81)

2736TMP70-89:
AAAAAAGGCTGGACTAAAGC

(SEQ ID NO: 82)

2736TMP372-393:
ACGTCAAAAAAATCATTATC

(SEQ ID NO: 83)

2736TMP619-647(GG):
GTTTTCGGTGCTCAAAAAGGGGCAAGTCC

(SEQ ID NO: 84)

2736TMP619-647(CC):
GTTTTCGGTGCTCAAAAAGGCGCAACTCC

Real-time PCR tests were carried out using the combinations of primers and a probe shown below in Table 24. 2736TMP619-647(GG) and 2736TMP619-647(CC) were used individually or as a mixture to provide a probe. In the table, the number in [ ] represents a SEQ ID NO. in SEQUENCE LISTING. The test bacterial strains used, the composition of the reaction liquid for the real-time PCR, and the reaction conditions were the same as in the above detection tests for the LMO0084 gene. When 2736TMP619-647(SS) was used, the reaction liquid composition was 0.25 μL of 100 μM 2736TMP619-647(GG) and 0.25 μL of 100 μM 2736TMP619-647(CC).

TABLE 25

LMO2736 primers
LMO2736

primer: F
primer: R
TaqMan (registered trademark) probe

1
F8 [32]
R176 [37]
2736TMP70-89 [81]

2
F222/M [70]
R591/M [74]
2736TMP372-393 [82]

3
F530/M [72]
R771/M [76]
2736TMP619-647(GG) [83]

4
F530/M [72]
R771/M [76]
2736TMP619-647(CC) [84]

5
F530/M [72]
R771/M [76]
2736TMP619-647(GG) [83]

2736TMP619-647(CC) [84]

The results are shown in Tables 26-1 to 26-3. The primer-probe sets 1 and 2 in Table 25 were capable of specific detection of the monocytogenes bacterium. 2736TMP619-647(GG) and 2736TMP619-647(CC) were capable of specific detection of the monocytogenes bacterium when they were used as a mixed probe. Based on the above results, 1, 2, and 5 in Table 25 are primer-probe combinations that can be especially preferably used for detection of the LMO2736 gene by real-time PCR.

TABLE 26-1

TaqMan ® probe

TMP70-89
TMP372-393
TMP619-647
TMP619-647
TMP619-647

−
−
GG
CC
Mix (GG/CC)

primer: F

F8
F222/M
F530/M
F530/M
F530/M

primer: R

R176
R591/M
R771/M
R771/M
R771/M

LMO 2736
size
168
368
241
241
241

1

Listeria

1/2a
GTC02947
+
+
+
−
+

monocytogenes

2

Listeria

1/2b
GTC02948
+
+
+
−
+

monocytogenes

3

Listeria

1/2c
JMC7672
+
+
+
−
+

monocytogenes

4

Listeria

3a
JMC7673
+
+
+
−
+

monocytogenes

5

Listeria

3b
JMC7677
+
+
−
+
+

monocytogenes

6

Listeria

3c
JMC7678
+
+
+
−
+

monocytogenes

7

Listeria

4a
JMC7674
+
+
−
+
+

monocytogenes

8

Listeria

4b
JMC7675
+
+
−
+
+

monocytogenes

9

Listeria

4d
JMC7680
+
+
−
+
+

monocytogenes

10

Listeria

5
GTC02957
+
+
−
+
+

monocytogenes

TABLE 26-2

TaqMan ® probe

TMP70-89
TMP372-393
TMP619-647
TMP619-647
TMP619-647

−
−
GG
CC
Mix (GG/CC)

primer: F

F8
F222/M
F530/M
F530/M
F530/M

primer: R

R176
R591/M
R771/M
R771/M
R771/M

LMO 2736
size
168
368
241
241
241

11

Listeria ivanovii

GTC02961
−
−
−
−
−

12

Listeria ivanovii

JMC7681
−
−
−
−
−

subsp. Ivanovii

13

Listeria ivanovii

GTC01640T
−
−
−
−
−

subsp. Ivanovii

14

Listeria ivanovii

GTC01641
−
−
−
−
−

subsp. londoniensisi

15

Listeria innocua

GTC16426T
−
−
−
−
−

16

Listeria innocua

GTC02960
−
−
−
−
−

17

Listeria welshimeri

GTC02963T
−
−
−
−
−

18

Listeria seeligeri

GTC16428T
−
−
−
−
−

19

Listeria grayi

GTC02964T
−
−
−
−
−

20

Listeria murrayi

GTC02964
−
−
−
−
−

21

Listeria marthii

GTC16430T
−
−
−
−
−

22

Listeria rocourtiae

GTC16429T
−
−
−
−
−

TABLE 26-3

TaqMan ® probe

TMP70-89
TMP372-393
TMP619-647
TMP619 647
TMP619-647

−
−
GG
CC
Mix (GG/CC)

primer: F

F8
F222/M
F530/M
F530/M
F530/M

primer: R

R176
R591/M
R771/M
R771/M
R771/M

LMO 2736
size
168
368
241
241
241

23

Escherichia coli (K12)
ATCC10798
−
−
−
−
−

24

Salmonella subsp.
JA.107
−
−
−
−
−

Enterica (I)

25

Salmonella subsp.
JA.125
−
−
−
−
−

Salamae (II)

26

Salmonella subsp.
JA.76
−
−
−
−
−

Arizonae (IIIa)

27

Salmonella subsp.
JA.129
−
−
−
−
−

Diarizinae (IIIb)

28

Salmonella subsp.
JA.n-22
−
−
−
−
−

Houtenae (IV)

29

Salmonella bongori (V)
JA.94
−
−
−
−
−

30

Salmonella subsp.
ATCC43971
−
−
−
−
−

EntericaTyphimurium

31

Staphylococcus aureus

ATCC6538P
−
−
−
−
−

32

Staphylococcus aureus

ATCC25923
−
−
−
−
−

33

Staphylococcus aureus

ATCC29213
−
−
−
−
−

34

Staphylococcus aureus

JMC2197
−
−
−
−
−

35

Staphylococcus aureus

IMCB.IMA2
−
−
−
−
−

36

Staphylococcus cohnii

ATCC29974
−
−
−
−
−

40

Staphylococcus saprophyticus

ATCC15305
−
−
−
−
−

37

Staphylococcus haemolyticus

ATCC29970
−
−
−
−
−

38

Staphylococcus hyicus subsp.
ATCC11249
−
−
−
−
−

39

Staphylococcus intermedius

ATCC29663
−
−
−
−
−

41

Citrobacter freundii

ATCC8090
−
−
−
−
−

Citrobacter freundii

N-326
−
−
−
−
−

43

Proteus vulgaris

IFO3988
−
−
−
−
−

44

Lactbacillus bulgarius

IFO13953
−
−
−
−
−

45

Lactbacillus helveticus

IFO3809
−
−
−
−
−

46

Streptcoccus sp.
IFO3535
−
−
−
−
−

47

Streptcoccus sanguis

ATCC10558
−
−
−
−
-

48

Streptcoccus mitis

ATCC6249
−
−
−
−
-

The following is an example of the procedure for preparation of a DNA sample in a case where a monocytogenes bacterium test is carried out for food using the present real-time PCR detection system.

(1) To 25 g of food, 225 mL of a bacterial growth selection medium is added, and culture is performed at 30° C. for 24 hours±3 hours. To 10 mL of BHI (Brain-Heart Infusion) medium, 0.1 mL of the resulting culture is added. Alternatively, a single colony on a selection agar medium is picked up, and then inoculated to 10 mL of BHI medium, followed by carrying out culture at 37° C. for 24 hours±3 hours.

(2) Centrifugation (13,000×g, 10 minutes, 20° C.) is carried out to collect bacterial cells from 1 mL of the resulting culture.

(3) DNA is extracted using a DNA extraction kit such as a mericon DNA Bacteria Plus Kit (QIAGEN: 69534).

(4) The DNA concentration is measured.

It was shown that, by this, monocytogenes can be specifically detected using a TaqMan (registered trademark) probe designed in the LMO0084 gene or the LMO2736 gene. By using a mixture of a plurality of TaqMan (registered trademark) probes taking polymorphic sequences of these genes into account, various serotypes of the monocytogenes bacterium can be comprehensively and specifically detected. The results shown in Table 22-1 and Table 26-1 indicate that, by designing a probe for targeting a polymorphism characteristic to a particular serotype, the monocytogenes bacterium can be detected specifically to the serotype, that is, serotype identification is possible. For example, 0084TMP535-558(CC) is a probe capable of specific detection of the serotype 4a. By designing new primers and probes from the regions in these two genes identified by the present inventors as target regions for specific detection of the monocytogenes bacterium, or from other regions, and appropriately combining these, identification of serotypes of the monocytogenes bacterium is possible.

LISTERIA-MONOCYTOGENES DETECTION METHOD

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