DETECTION OF METHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS

BACKGROUND OF THE INVENTION

Methicillin-resistant Staphylococcus aureus (MRSA) strains contain a SCCmec cassette, which may contain the mecA gene whose gene product confers methicillin resistance. Specific detection of MRSA by the presence of the SCCmec cassette has been used in a number of publications and patents (U.S. Pat. No. 6,156,507, US 2005019893). These various methods all rely on the use of primer set(s) where one (or more) primers target the S. aureus chromosomal DNA (external to SCCmec) and the other one (or more) primers target the SCCmec cassettes near the chromosomal integration site. Due to the presence of multiple SCCmec (sub) types, more than one primer set is needed to detect all MRSAs. SCCmec cassettes are also found in other Staphylococcus species, commonly known as coagulase-negative staphylococci, including S. epidermidis, S. hominis, S. haemolyticus and others. Current methods therefore use primers directed specifically to S. aureus chromosomal DNA (non-cassette regions) to assess the species identification and to avoid confusion between SCCmec cassettes detected in S. aureus and those in other staphylococci.

In theory, this is a clear strategy for MRSA detection, however, in practice this concept produces both false positive and false negative results as has been reported (Drews, et. al., J. Clin. Microbiol. 44:3794, 2006; Desjardins, et. al., J. Clin. Microbiol. 44:1219, 2006, Bishop, et. al. J. Clin. Microbiol. 44:2904, 2006) and there is therefore a need for improved methods with higher sensitivity and specificity.

SUMMARY OF THE INVENTION

Oligonucleotides or oligonucleotide mimics as defined herein can be used in methods to detect MRSA and MRCNS and to distinguish MRSA from MRCNS. The oligonucleotides and oligonucleotide mimics are, in some embodiments, set forth in the tables herein, and comprise sequences SEQ ID NOs 1-36. The oligonucleotides and oligonucleotide mimics are generally referred to as probes, especially as used in hybridization methods, or as primers, especially as used in DNA amplification methods.

In one embodiment the invention is a method of testing a sample for the presence or absence of SCCmec cassette DNA present as an insertion in chromosomal DNA of Staphylococcus aureus. The method includes (a) hybridizing the sample, under appropriate conditions, to one or more capture probe(s) that hybridize to chromosomal DNA of Staphylococcus aureus external to SCCmec by at least partially complementary sequences, the capture probe(s) being optionally attached to a support, (b) hybridizing the sample to one or more detector probe(s) that hybridize to SCCmec cassette DNA by at least partially complementary sequences, and (c) detecting each of the detector probe(s), which may include application of means of detection appropriate to the probe (and not all probes may be detected) thereby determining that the SCCmec cassette DNA is present and that it is integrated into the chromosomal DNA of Staphylococcus aureus. The hybridizations (a) and (b) can be carried out at the same time or can be carried out in either order. Optionally, the sample to be tested can be hybridized to one or more blocker probe(s) that hybridize to DNA of one or more other species of staphylococci as part of step (a).

Another method tests a sample for the presence of DNA of S. aureus comprising SCCmec. The method includes performing an amplification reaction using the sample as a source of template DNA. A first primer comprises a nucleobase sequence complementary to a nucleobase sequence within SCCmec of at least one SCCmec type of Staphylococcus species. A second primer hybridizes to chromosomal DNA of S. aureus external to SCCmec. The results of the amplification reaction are analyzed for the presence of amplification product. If the amplification product is present, DNA of S. aureus comprising SCCmec is present in the sample.

Further aspects of the invention are kits with components to carry out the assay methods. The kits include oligonucleotide or oligonucleotide mimics to be used as probes or primers, as appropriate to the method, and can include other reagents and solutions. A solid support may be supplied with capture probe attached, or for attachment of capture probes, for example.

DETAILED DESCRIPTION OF THE INVENTION

The design of S. aureus-specific probes near the integration site for the SCCmec cassette is complicated by the high degree of sequence similarity between the chromosomal DNA of Staphylococcus species, (herein, the term “chromosomal DNA” is limited to regions external to any SCCmec, if present) which may explain false-positive results by current methods (Francois, et al., J Clin Microbiol. 45(6):2011-2013, 2007). With this invention, improved specificity is possible using blocker probes that prevent hybridization of S. aureus-specific probes to other Staphylococcus species.

Another parameter complicating probe design is sequence heterogeneity of chromosomal DNA between S. aureus strains within the S. aureus-specific target regions, such that a single probe does not hybridize to all S. aureus strains as shown in Example 3. This may be overcome by simply designing multiple probes as also shown in Example 3, but this invention offers the advantage of designing fewer probes, or even a single probe directed towards a non-S. aureus-specific region which, when used in conjunction with blocker probes as shown in Example 6, eliminates hybridization to other Staphylococcus species.

The invention includes oligonucleotides and oligonucleotide mimics, including those comprising the nucleobase sequences described herein, and including those consisting of the nucleobase sequences described herein. Probes and primers comprising oligonucleotides or oligonucleotide mimics are part of the invention and are suitable for the assays described herein. Oligonucleotide mimics include, for example, phosphorothioate oligonucleotides, peptide nucleic acids (PNAs), and locked nucleic acids (LNAs). Oligonucleotide mimic portions and oligonucleotide portions (comprising at least one monomer unit, for example) can be combined in one chimeric oligonucleotide (e.g., a PNA/DNA chimera or a LNA/DNA chimera). Such chimeric oligonucleotides are also included within “oligonucleotide mimics.” Like oligonucleotides that may be found naturally occurring in cells, oligonucleotide mimics are spoken of as having a nucleobase sequence according to the A, C, G, T and U base portion of each of their respective monomer units. Also like oligonucleotides that may be found naturally occurring in cells, oligonucleotide mimics can form hybrids through Watson-Crick basepairing with DNA and/or RNA comprising a nucleobase sequence that is at least partially complementary. The oligonucleotides may be isolated. An oligonucleotide or oligonucleotide mimic can be provided as (in solution for example) the only probe, or it can be provided as a combination of one or more probes.

Generic probes are presented which bind to conserved regions of nucleic acid targets, though with some mismatched bases. As described herein, probes were designed that can form hybrids with these regions even though many of the bases in the probe are mismatched. The probe design is based on an “average” sequence from the alignment of several sequences derived from nature. The probe incorporates bases at each position which are complimentary to at least one of the natural sequences. The resulting probe is partially complimentary to all of the sequences it was designed against. The design method is similar to the well known concept of degenerate probes (primers), where a given probe sequence is designed with “wobble” bases incorporated, such that the final probe produced is actually composed of a population of two or more distinct sequences based on a general core sequence. This design method is different from degenerate probes in that there is only one probe sequence based on consensus complementarities. This type of generic mismatching probe is referred to as a “consensus” probe herein.

Consensus probes can be used in combination with blocker probes. Blocker probes are well defined in the art and at their simplest can be defined as probes which bind specifically to nucleic acid targets which are not targets of interest. Very often blocker probes are designed such that they can not be detected. Blocker probes are used as part of a probe cocktail to prevent evolution of detectable signal from targets which are not of interest. Blocker probes and consensus probes together, as described herein, can be used to prevent signal evolution from specific nucleic acid targets.

The combination of blocker probes and consensus probes (with either partially or complete complementarity to the target and/or non target sequence) offers the advantage of utilizing sequence differences outside the target/non-target region, such that only a part of the blocker probe(s) is used to reduce binding of the consensus probe to the non-target sequence. The use of one and two blocker probes utilizing sequence differences is exemplified in Example 5 and 6. This aspect of the invention is not limited to the sample or assay type but is a general concept providing improved specificity for nucleic acid analysis, and can be viewed as a means of using a longer target sequence.

Another aspect of this invention is the use of very short probes which facilitates design of probes outside target regions with sequence heterogeneity. Use of very short probes, i.e. less than 10 bases, is common in the art for applications where specificity is not desired, i.e., random priming of PCR reactions with hexamers, as any given 9-mer is statistically predicted to occur every 4 to the 9^th(4⁹=262144) bases. Very short probes are therefore not perceived by prior art in nucleic acid detection methods where high specificity is required, and in fact were specifically ruled out by earlier practitioners as being untenable for detection of SCCmec cassettes in S. aureus (US 2005019893). This exclusion may have been due to the reliance on PCR as a dominant method of nucleic acid analysis. As shown herein, very short probes containing LNA and/or PNA allow for nucleic acid detection methods, which can not be performed using conventional DNA probes of equivalent length (see Examples 4 and 5). The advantages of using short probes are not limited to the sample or assay type.

This invention is exemplified for the detection of MRSA but is equally suitable for detection of MRCNS and in fact offers the potential for using the same probe set(s) comprising consensus probe(s) and probe(s) for SCCmec for detection of the SCCmec cassette in staphylococci (with differentiation between MRSA and MRCNS (methicillin-resistant coagulase negative)) and supplemented with either blocking probes preventing hybridization of the consensus probe to non-S. aureus staphylococci for detection of MRSA, or blocking probes preventing hybridization of the consensus probe to S. aureus for detection of MRCNS.

In one embodiment, the invention comprises probes for the detection of SCCmec in S. aureus; see Examples 1, 2 and 3. Preferably, the probes comprise less than 15 nucleobases and are oligonucleotide mimics comprising LNA or PNA, detecting one or more SCCmec types (see Examples 4 and 5) and may be used in combination to detect multiple SCCmec types (see Example 4 and Example 5).

In another embodiment, the invention comprises a method for detecting SCCmec in S. aureus using the probes in combination with one or more probes hybridizing to chromosomal DNA of S. aureus. These may be either hybridizing specifically to S. aureus only (Example 6) or to other species of Staphylococci (Example 7). To eliminate hybridization to other Staphylococcus species, blocker probes may be included to prevent hybridization of the probe(s) to other Staphylococcus species (Examples 7 and 8). Steps of methods for specific purposes are described herein, wherein step a) (hybridizing the sample to one or more capture probes and step b) (hybridizing the sample to one or more detector probes) can occur by combining the appropriate reagents in sequence, or can occur by combining the appropriate reagents in one hybridization mixture so that hybridizing steps a) and b) occur at the same time.

The method to detect SCCmec in S. aureus in a sample may include lysing the cells in the sample, denaturing the DNA in the sample, incubating the DNA with one or more capture probes hybridizing to SCCmec types and one or more probes hybridizing to S. aureus chromosomal DNA. Protocols to carry out sandwich-type DNA hybridization assays or target amplification assays are known in the art.

Kits comprising probe set(s) and optionally also comprising reagents are also a part of the invention. Instructions to use the kit can also be a component of the kit. DNA control standards can also be included. Capture probes can be supplied attached to a support, such as in the wells of a microtiter plate. One capture probe can be supplied in a set of wells, or more than one capture probe can be supplied together in the same wells. Detector probes can be provided individually packaged, or more than one detector probe can be packaged together. Blocker probes, if included in the kit, can be provided individually packaged, or more than one blocker probe can be packaged together. Blocker probes can be included with detector probes. It will be understood that while certain probes that hybridize to a first region of DNA are designated herein as “capture” probes and certain probes that hybridize to a second region of DNA are designated herein as “detector” probes, probes of the sequences of capture probes can be used in assays as detector probes and vice versa, so long as the appropriate probes are used together in the assay.

This invention is exemplified using sandwich hybridization but is also applicable to other hybridization methods and to nucleic acid amplification techniques.

DNA amplification methods (e.g., polymerase chain reaction) can be used to detect MRSA in a sample, as these methods, in effect, detect a nucleobase sequence internal to SCCmec, and a nucleobase sequence on the S. aureus chromosome external to SCCmec on a template DNA of S. aureus present in the sample. The amplification methods can use as primers any of the oligonucleotides or oligonucleotide mimics described as probes herein, or can use shorter portions of those molecules. Primers of a variety of lengths, comprising PNA or LNA, can be used, e.g., primers of 14, 13, 12 11, or fewer monomers. The primers can comprise the nucleobase sequences described for any of the oligonucleotides or oligonucleotide mimics described herein as capture probes or detector probes, so that a primer pair for amplification consists of one oligonucleotide or oligonucleotide mimic with the nucleobase sequence of a capture probe and one oligonucleotide or oligonucleotide mimic with the nucleobase sequence of a detector probe.

It should be noted from the results in Example 12 that LNA/DNA primers (comprising both LNA and DNA monomers) shorter than DNA primers of the same sequence can be used successfully to amplify DNA by the polymerase chain reaction. LNA/DNA primers as short as 10 monomer units resulted in amplified product.

The detection of MRSA by sandwich hybridization methods and by amplification methods such as the methods shown herein exemplifies a more general method that can be applied to the detection of other DNA or RNA targets (e.g., a gene for virulence in pathogenic bacteria, an allele associated with a genetic disease in humans, or a sequence indicating a mutation, a chromosomal rearrangement, deletion or insertion). The use of short LNA or LNA/DNA oligonucleotide mimics as probes or primers as demonstrated herein (see, for example, Examples 11 and 12) can be generally applied to the detection of other target DNAs in other assays. Oligonucleotide mimics comprising LNA can be substituted for oligonucleotides of the same nucleobase sequence for use as primers or probes, with greater sensitivity in assays. The length of an effective probe or primer can be shorter in an oligonucleotide mimic comprising LNA, compared to an oligonucleotide.

As used herein, the word “hybridize” or “hybridizing” means the sequence specific binding of any of two single stranded nucleic acids or oligonucleotides or oligonucleotide mimics to form duplexes according to Watson-Crick base-pairing rules. Hybrids may form between two nucleobase sequences which are not perfectly complementary as defined by the W-C base pairing rules, but are substantially complimentary such that they form stable double stranded complexes under assay conditions, also referred to as partially complementary consensus probes.

A sample to be used in the assays described herein can be, for example, bacteria from one or more isolated colonies, or bacteria grown in a liquid or other culture, either isolated or mixed. Additionally, a sample can be bodily fluid or washings as might be obtained for analysis in a clinical laboratory, or an aliquot of such fluid or washings.

Staphylococcus species is used to indicate any of the species of staphyloccocci.

EXAMPLES
Materials and Methods

The examples below use EVIGENE, a sandwich-type DNA hybridization assay (Skov et al., Journal of Antimicrobial Chemotherapy 43: 467-475, 1999; Levi and Towner, J. Clin. Microbiol. 38: 830-838, 2003) to specifically detect genetic markers in staphylococci. EVIGENE kit components (AdvanDx, Woburn, Mass.) and generic assay products were used for all experiments differing only by the individual probes as listed in the Examples below. For each sample, 2 drops of Lysis I solution were added to 2 mL microcentrifuge tubes with lockable lids. Using a 1 μL plastic inoculating loop, 2 loops of colonies were collected and transferred to the tube and vortexed until the bacteria were completely suspended. The tube was incubated in a heating block or water bath at 35-37° C. for 20 min. Two drops of Lysis II solution were added to the tube and vortexed thoroughly. The tube was incubated in a heating block at 95-100° C. for 15 min, vortexed and incubated at 95-100° C. for another 15 min. The tubes were allowed to cool at room temperature for 5 min. One drop of Detector Probes was added into a well in a microtiter well with immobilized Capture Probes followed by 100 μL of the sample (lysed bacteria) from the tube. The resulting mixture comprised NaCl, Tris and detergent as known to facilitate hybridization of the probes to the bacterial nucleic acid. The well was covered with plate sealing tape and placed in a microtiter plate shaker/incubator set at 55° C. and 400 rpm and incubated for 60 min. After incubation, the well was emptied and 2 drops of Conjugate Buffer were added, followed by 1 drop of Conjugate. The well was covered with plate sealing tape and incubated at 35-37° C. for 30 min. After the end of incubation, the well was then emptied, washed 4 times with 7 drops (or 4×200 μl) of Wash Solution. One drop of Substrate A was added followed by 2 drops of Substrate B and the solution was incubated at room temperature for 30 min. Two drops of Stop Solution were added and absorbance was measured at 492 nm using an ELISA plate reader. Other assay formats, including PCR or other target amplification techniques, may also be used with the probe sets.

A variety of bacteria strains from AdvanDx A/S, Denmark were used. The identity (species and type, if available) of each isolate was based on information available from the supplier of the strains and/or other tests, such as EVIGENE tests for detection of mecA (gene for methicillin resistance) and nuc (S. aureus-specific gene).

DNA probes and probes comprising LNA nucleobases were obtained from TAG Copenhagen A/S (Copenhagen, Denmark). Capture probes were labeled for immobilization onto microtiter wells and Detector probes were labeled for detection via colorimetric signal amplification using components from commercial EVIGENE kits (AdvanDx). Blocker probes were unlabeled.

Example 1
Detection of SCCmec Type I, II, and IV in S. aureus, Only

Table 1.1 displays the nucleobase sequences of the Capture and Detector probes used in the experiment. The Capture probe targets the S. aureus DNA and has 10 mismatches to Staphylococcus epidermidis DNA. The Detector probe targets SCCmec types I, II, and IV (not III and V).

TABLE 1.1

Nucleobase Sequences

Probe

Type
Name
Sequence (5′ to 3′)

Capture
GenCP2
AATCCTTCGGAAGATAGCATCTTTCCTTGTATTTCT

AATG

(SEQ ID NO: 1)

Detector
Dt1
GCTATTATTTACTTGAAATGAAAGACTGCGGAGGCT

AACT

(SEQ ID NO: 2)

Method

Samples were prepared and assayed essentially according to the EVIGENE™ procedure using Capture probe GenCP2 (60 nM) and Detector Probe Dt1 (5 nM).

Results

The mecA result of each isolate (using the MRSA EVIGENE™ kits) as well as the MRSA results according to the invention, i.e. specific detection of mecA in S. aureus by detection of the SCCmec cassette in S. aureus only, are shown in Table 1.2. The final column displays the absorbance readings at 492 nm. Absorbance readings above background levels indicate the presence of the nucleic acid target in the sample.

TABLE 1.2

Results of Example 1

Species
ATCC#
Type
MecA
MRSA
OD 492

Staphylococcus aureus

6538
MecA neg.
Negative
Negative
0.287

Stphylococcus epidermis

51625
CNS
Positive
Negative
0.153

Enterococcus faecalis

51299

Negative
Negative
0.122

Enterococcus faecium

700221

Negative
Negative
0.116

Staphylococcus aureus

SCCmec type I
Positive
Positive
1.231

Staphylococcus aureus

SCCmec type Ia
Positive
Positive
1.049

Staphylococcus aureus

SCCmec type II
Positive
Positive
1.121

Staphylococcus aureus

SCC mec type II
Positive
Negative
0.185

Staphylococcus aureus

SCCmec type IIa
Positive
Negative
0.215

Staphylococcus aureus

SCCmec type IV
Positive
Positive
1.033

Staphylococcus aureus

SCCmec type IV - PV_pos. ST80
Positive
Positive
1.261

Staphylococcus epidermis

CNS
Positive
Negative
0.171

Unknown

CNS
Positive
Negative
0.165

Staphylococcus haemolyticus

CNS
Positive
Negative
0.244

Staphylococcus simulans

27851
CNS
Negative
Negative
0.167

Staphylococcus warneri

49454
CNS
Negative
Negative
0.178

Staphylococcus aureus

Negative
Negative
0.135

Staphylococcus aureus

Negative
Negative
0.139

Staphylococcus fleurettii

BAA-274
CNS
Positive
Negative
0.436

Staphylococcus

Positive
Negative
0.205

Staphylococcus saccharolyticus

14953
CNS
Positive
Negative
0.192

Staphylococcus aureus

700699
MecA pos.
Positive
Positive
0.673

Staphylococcus aureus

MecA pos.
Positive
Positive
0.974

Staphylococcus aureus

MecA pos.
Negative
Negative
0.140

Escherichia coli

35218

Negative
Negative
0.184

Candida krusei

Y-7550

Negative
Negative
0.120

Staphylococcus aureus

SCCmec type I
Positive
Positive
1.628

Staphylococcus aureus

SCCmec type Ia
Positive
Positive
1.328

Staphylococcus aureus

SCCmec type I
Positive
Positive
1.297

Staphylococcus aureus

SCCmec IA - MecA pos.
Positive
Positive
0.614

Staphylococcus aureus

SCCmec type II
Positive
Positive
1.315

Staphylococcus aureus

SCCmec type II
Positive
Positive
1.204

Staphylococcus aureus

SCCmec type II
Positive
Positive
1.402

Staphylococcus aureus

SCCmec type IIIa
Positive
Negative
0.211

Staphylococcus aureus

SCCmec type IIIa
Positive
Negative
0.142

Staphylococcus aureus

SCCmec type III
Positive
Negative
0.133

Staphylococcus aureus

SCCmec type IV
Positive
Positive
1.324

Staphylococcus aureus

SCCmec type III
Positive
Negative
0.172

Staphylococcus aureus

SCC mec type V
Positive
Negative
0.141

Staphylococcus aureus

SCCmec type IV
Positive
Positive
1.306

Staphylococcus aureus

Spa type 10$$5
Positive
Positive
1.300

Staphylococcus aureus

Spa type 1044
Positive
Positive
1.345

Staphylococcus aureus

Spa type 1175
Positive
Positive
1.021

Staphylococcus aureus

Spa type 1019
Positive
Positive
1.420

Unknown
SSI B
CNS - MecA/nuc pos.
Positive
Negative
0.230

The results clearly show that only mecA-positive S. aureus strains of SCCmec type I, II or IV tested positive (OD₄₉₂>0.5); whereas, mecA-positive staphylococci other than S. aureus, mecA-negative S. aureus and other bacterial and yeast strains all tested negative (OD₄₉₂<0.5). The results also showed that mecA-positive S. aureus strains of SCCmec type III and V tested negative, as the Detector probe was not designed for these SCCmec types.

The data also show that sandwich hybridization, such as EVIGENE, is a suitable assay format and is thus an alternative to PCR for specific detection of SCCmec in S. aureus.

Example 2
Detection of SCCmec Type I, II, III, IV and V in Only S. Aureus

Table 2.1 displays the nucleobase sequences of the Capture and Detector probes used in the experiment. Capture probe GenCP2 (the same as in Example 1). Detector probe Dt1 (designed to be specific for SCCmec type I, II & IV), Detector probe Type III (designed to be specific for SCCmec type III) and Detector probe Type V (designed to specific for SCCmec type V, Table 2.1).

TABLE 2.1

Nucleobase Sequences

Probe

Type
Name
Sequence (5′ to 3′)

Capture
GenCP2
AATCCTTCGGAAGATAGCATCTTTCCTTGTATTT

CTAATG

(SEQ ID NO: 1)

Detector
Dt1
GCTATTATTTACTTGAAATGAAAGACTGCGGAGG

CTAACT

(SEQ ID NO: 2)

Detector
Type III
TAATCCAATATTTCATATATGTAATTCCTCCACA

TCTCAT

SEQ ID NO: 3)

Detector
Type V
ACTTTAGTCAAATCATCTTCACTAGTGTAATTAT

CGAATG

(SEQ ID NO: 4)

Method

Samples were prepared and assayed essentially according to the EVIGENE™ procedure using Capture probe GenCP2 (60 nM) and Detector Probe Dt1 (5 nM). Detector Probe Type III (5 nM), or Detector Probe Type V (5 nM).

TABLE 2.2

Results of Example 2

Capture probe GenCp2

AdvanDx

Dt1 (Type I,

OD

Species
ID
Type
II, IV)
OD 492
Type III
OD 492
Type V
492

Staphylococcus aureus

#294
SCCmec type I
Positive
1.628
Negative
0.179
Negative
0.194

Staphylococcus aureus

#296
SCCmec type I
Positive
1.297
Negative
0.166
Negative
0.174

Staphylococcus aureus

#295
SCCmec type Ia
Positive
1.328
Negative
0.192
Negative
0.185

Staphylococcus aureus

#298
SCCmec type II
Positive
1.316
Negative
0.186
Negative
0.204

Staphylococcus aureus

#299
SCCmec type II
Positive
1.204
Negative
0.203
Negative
0.178

Staphylococcus aureus

#300
SCCmec type II
Positive
1.402
Negative
0.172
Negative
0.157

Staphylococcus aureus

#301
SCCmec type IIIa
Negative
0.211
Positive
1.255
Negative
0.178

Staphylococcus aureus

#303
SCCmec type III
Negative
0.133
Positive
1.297
Negative
0.198

Staphylococcus aureus

#305
SCCmec type III
Negative
0.172
Positive
1.280
Negative
0.202

Staphylococcus aureus

#304
SCCmec type IV
Positive
1.324
Negative
0.158
Negative
0.171

Staphylococcus aureus

#307
SCCmec type IV - PVL
Positive
1.306
Negative
0.216
Negative
0.351

pos.

Staphylococcus aureus

#460
Type V
Negative
0.250
Negative
0.232
Positive
1.091

Staphylococcus

#351
MR-CNS
Negative
0.160
Negative
0.139
Negative
0.224

warneri

Staphylococcus

#352
MR-CNS
Negative
0.166
Negative
0.164
Negative
0.330

epidermis

Staphylococcus

#353
MR-CNS
Negative
0.185
Negative
0.142
Positive
0.578

hominis

Results

The results show that only MRSA strains typed as SCCmec type I, II & IV tested positive (OD₄₉₂>0.5) with Detector Dt1 and strains typed as SCCmec type III tested positive (OD₄₉₂>0.5) with Detector Type III and strains typed as SCCmec type V tested positive (OD₄₉₂>0.5) with Detector Type V; whereas, mecA-positive staphylococci other than S. aureus, all tested negative (OD₄₉₂<0.5).

Example 3

Table 3.1 displays the nucleobase sequences of the Capture and Detector probes used in the experiment. Additional detector probes were tested based on sequences in WO02099034. Two Capture probes GenCP2 (used in previous Examples) and GenCP3 were tested with Detector probes Dt1, Type III, Type V, Type5 (specific for SCCmec type V, WO02099034), Type4 (specific for SCCmec type IV, WO02099034), Type7 (specific for SCCmec type VII, WO02099034), type 6 (specific for SCCmec type VI, WO02099034), or type 8,9 (designed to be specific for SCCmec types VIII and IX, WO02099034).

TABLE 3.1

Nucleobase Sequences

Probe

Type
Name
Sequence (5′ to 3′)

Capture
GenCP2
AATCCTTCGGAAGATAGCATCTTTCCTTGTATT

TCTAATG

(SEQ ID NO: 1)

Capture
GenCP3
TGTTCAATTAACACAACCCGCATCATTTGATGT

GGGAATG

(SEQ ID NO: 5)

Detector
Dt1
GCTATTATTTACTTGAAATGAAAGACTGCGGAG

GCTAACT

(SEQ ID NO: 2)

Detector
Type III
TAATCCAATATTTCATATATGTAATTCCTCCAC

ATCTCAT

(SEQ ID NO: 3)

Detector
Type V
ACTTTAGTCAAATCATCTTCACTAGTGTAATTA

TCGAATG

(SEQ ID NO: 4)

Detector
Type5
TAATAAACTCTGCTTTATATTATAAAATTACGG

CTGAAATAACC

(SEQ ID NO: 6)

Detector
Type4
TGTTTTCTTCAAATATTATCTCGTAATTTACCT

TGTTCATTAAAC

(SEQ ID NO: 7)

Detector
Type7
TTTATTCTTCAAAGATTTGAGCTAATTTAATAA

TTTTCTCATA

(SEQ ID NO: 8)

Detector
type 6
AATTAATCTGATTTTCACTCTTCATCTACTTAC

TCATATT

(SEQ ID NO: 9)

Detector
type 8,
AACATAACAGCAATTCACATAAACCTCATATGT

9
TCTGATA

(SEQ ID NO: 10)

Method

Samples were prepared and assayed essentially according to the EVIGENE™ procedure using Capture probe GenCP2 (60 nM) and Detector probe Dt1 (5 nM) and Detector probe Type III (5 nM), or Detector probe Type V (5 nM), Detector probe Type5 (5 nM), Detector probe Type4 (5 nM), Detector probe Type7 (5 nM), Detector probe type 6 (5 nM), or Detector probe type 8,9 (5 nM). The Capture probe GenCP3 was tested together with Detector probe Dt1, Type III, and Type V (5 nM each) or with Detector probe Type5 (5 nM), Detector probe Type4 (5 nM), Detector probe Type7 (5 nM), Detector probe type 6 (5 nM), or Detector probe type 8,9 (5 nM).

TABLE 3.2

Results of Example 3

Capture probe GenCp2

Dt1 &
OD

OD

OD

OD

OD

OD

OD

Sample
ID
Type III
492
Type V
492
Type 5
492
Type 4
492
Type 7
492
Type 6
492
Type 8, 9
492

Clinical
#360
Negative
0.155
Negative
0.115
Negative
0.264
Negative
0.216
Negative
0.185
Negative
0.214
Positive
1.672

isolate

Clinical
#361
Negative
0.159
Negative
0.124
Negative
0.290
Negative
0.226
Negative
0.267
Negative
0.201
Positive
1.769

isolate

Clinical
#389
Negative
0.121
Negative
0.111
Negative
0.226
Positive
1.578
Negative
0.205
Negative
0.153
Negative
0.172

isolate

Clinical
#391
Negative
0.135
Negative
0.124
Positive
1.947
Negative
0.236
Negative
0.251
Negative
0.204
Negative
0.179

isolate

Clinical
#395
Negative
0.141
Negative

Negative
0.212
Negative
0.183
Positive
1.711
Negative
0.146
Negative
0.243

isolate

Clinical
#396
Negative
0.129
Negative
0.116
Positive
1.703
Negative
0.218
Negative
0.223
Negative
0.195
Negative
0.226

isolate

Clinical
#397
Negative
0.136
Negative
0.109
Negative
0.229
Negative
0.230
Negative
0.273
Negative
0.185
Negative
0.239

isolate

Capture probe GenCp3

Dt1 &

Type III &
OD

OD

OD

OD

OD

OD

Sample
ID
Type V
492
Type 5
492
Type 4
492
Type 7
492
Type 6
492
Type 8, 9
492

Clinical
#397
Negative
0.130
Negative
0.124
Negative
0.105
Negative
0.119
Positive
1.840
Negative
0.105

isolate

Results

The results in Example 3 show that the strains which did not test positive with GenCp2 and the detector probes described in Example 2 (Dt1, Type III & Type V) tested positive when alternate detector probes or another capture probe (GenCp3) was included in the test. The data show that multiple Capture and Detector probes may improve sensitivity and that additional probes may be added as additional “false-negative” strains (new SCCmec types) occur.

Example 4
Detection of mecA in Only S. aureus by Detection of the SCCmec Cassette in S. aureus with 9-mer LNA Probe

Table 4.1 displays the nucleobase sequences of the Capture and Detector probes used in the experiment.

TABLE 4.1

Nucleobase Sequences

Probe

Type
Name
Sequence (5′ to 3′)

Capture
GenCP3
TGTTCAATTAACACAACCCGCATCATTTG

ATGTGGGAATG

(SEQ ID NO: 5)

Detector
Dt1
GCTATTATTTACTTGAAATGAAAGACTGC

GGAGGCTAACT

(SEQ ID NO: 2)

Detector
T1, 2, 4-Dt5
GaggctaaC

(SEQ ID NO: 11)

Detector
T1, 2, 4-Dt8
GAGGCTAAC

(SEQ ID NO: 12)

LNA monomers are lowercase a, g, c, t, and DNA monomers are uppercase A, G, C, T

Method

Samples were prepared and assayed essentially according to the EVIGENE™ procedure using the Detector probe Dt1(5 nM), 9-mer Dt5 LNA/DNA (50 nM) or 9-mer DNA (50 nM) and Capture probe GenCp3 (60 nM) above (Table 4.1).

TABLE 4.2

Results of Example 4

Dt5 (Type I,

Dt8 (Type

Dt1 (Type

II, IV) 9-mer

I, II, IV)

Species
ADx ID
Type
I, II, IV)
OD 492
LNA/DNA
OD 492
9-mer DNA
OD 492

Staphylococcus aureus

#294
SCCmec type I
Positive
2.475
Positive
1.713
Negative
0.123

Staphylococcus aureus

#295
SCCmec type Ia
Positive
2.398
Negative
0.469
Negative
0.112

Staphylococcus aureus

#298
SCCmec type II
Positive
2.766
Positive
1.992
Negative
0.164

Staphylococcus aureus

#303
SCCmec type III
Negative
0.112
Negative
0.111
Negative
0.118

Staphylococcus aureus

#301
SCCmec type IIIa
Negative
0.118
Negative
0.095
Negative
0.138

Staphylococcus aureus

#304
SCCmec type IV
Positive
2.981
Positive
1.584
Negative
0.125

Staphylococcus aureus

#307
SCCmec type IV + PVL
Positive
2.783
Positive
1.694
Negative
0.121

Staphylococcus aureus

#460
Type V
Negative
0.116
Negative
0.098
Negative
0.127

Staphylococcus warneri

#351
MR-CNS
Negative
0.389
Negative
0.169
Negative
0.131

Staphylococcus

#352
MR-CNS
Negative
0.129
Negative
0.101
Negative
0.124

epidermidis

Staphylococcus hominis

#353
MR-CNS
Negative
0.116
Negative
0.097
Negative
0.175

Staphylococcus aureus

#156
MSSA
Negative
0.112
Negative
0.101
Negative
0.114

Results

Results of Example 4 are summarized in Table 4.2, which displays the absorbance values obtained at 492 nm and the positive/negative results for the samples. The experiment showed that only the methicillin-resistant S. aureus strains of SCCmec type I, II or IV were positive (OD₄₉₂>0.5) using the Capture GenCp3 and a 40-mer DNA or a LNA/DNA 9-mer Detector Probe, whereas a 9-mer DNA Detector Probe with the same sequence as the LNA/DNA gave a negative result when testing the same samples.

The data also show that replacing some of the naturally occurring nucleotides with non-naturally-occurring LNA monomers allows for use of short probes.

Example 5
Detection of mecA in Only S. aureus by Detection of the SCCmec Cassette in S. aureus with 9-mer PNA Probe

Table 5.1 displays the nucleobase sequences of the Capture and Detector probes used in the experiment. The 9-mer PNA Detector Probe Dt10 was designed to detect SCCmec type I, II and IV.

TABLE 5.1

Nucleobase Sequences

Probe

Type
Name
Sequence (5′ to 3′)

Capture
GenCP2
AATCCTTCGGAAGATAGCATCTTTCCTTGTAT

TTCTAATG

(SEQ ID NO: 1)

Detector
T1, 2, 4-
ctgcggagg

Dt 10
(SEQ ID NO: 13)

PNA monomers are lowercase a, g, c, t, and DNA monomers are uppercase A, G, C, T

Method

Samples were prepared and assayed essentially according to the EVIGENE™ procedure using the Detector probe 9-mer Dt10 PNA (50 nM) and Capture probe GenCp2 (60 nM) above (Table 5.1).

Results

TABLE 5.2

Results of Example 5

Dt10 (Type I,

II, IV)

9-mer PNA

Species
ADx ID
Type
OD 492

Staphylococcus aureus

#294
SCCmec type I
Positive
0.659

Staphylococcus aureus

#295
SCCmec type Ia
Positive
0.674

Staphylococcus aureus

#297
SCCmec type Ia
Positive
0.535

Staphylococcus aureus

#298
SCCmec type II
Positive
0.797

Staphylococcus aureus

#303
SCCmec type III
Negative
0.230

Staphylococcus aureus

#301
SCCmec type IIIa
Negative
0.290

Staphylococcus aureus

#304
SCCmec type IV
Positive
0.651

Staphylococcus aureus

#460
Type V
Negative
0.200

Staphylococcus

#351
MR-CNS
Negative
0.198

warneri

Staphylococcus

#352
MR-CNS
Negative
0.194

epidermidis

Staphylococcus

#353
MR-CNS
Negative
0.177

hominis

Staphylococcus aureus

#156
MSSA
Negative
0.191

Results of Example 5 are summarized in Table 5.2, which displays the absorbance values obtained at 492 nm and the positive/negative results for the samples. The experiment showed that only the methicillin-resistant S. aureus strains of SCCmec type I, II or IV were positive (OD₄₉₂>0.5) using the Capture GenCp2 and PNA 9-mer Detector Probe (Dt10). The data also show that replacing some of the naturally occurring nucleotides with non-naturally-occurring PNA monomers allows for the use of short probes.

Example 6
Specific Detection of MRSA with S. warneri Blocker Probes

Table 6.1 displays the nucleobase sequences of the Capture and Detector probes used in the experiment.

In the experiment below, Capture probe (GenCp3, Table 4.1), all the SCCmec Detector probes (Dt1, type III, type V, Type5, Type4, Type7, type 6 and type 8,9, Table 3.1) and Blocker probes (B4A and B4B or the B5, Table 6.1) were used.

TABLE 6.1

Nucleobase Sequences

Probe

Type
Name
Sequence (5′ to 3′)

Capture
GenCP3
TGTTCAATTAACACAACCCGCATCATTTGATGTGGG

AATG

(SEQ ID NO: 5)

Detector
A11
See note*

Blocker
B4A
ATTTGGTGTGGGAAGGTCATCTTGCTGAA

(SEQ ID NO: 14)

Blocker
B4B
TCGATATACTTGTTCTATCAACACGACGCG

(SEQ ID NO: 15)

Blocker
B5
TGTTCTATCAACACGACGCGCATCATTTGG

TGTGGGAAGG

(SEQ ID NO: 16)

* All Dt SCCmec: Dt1, type III, type V, Type5, Type4, Type7, type 6 and type 8, 9 (see Example 3 for the sequences)

Method

Samples were prepared and assayed essentially according to the EVIGENE™ procedure using the Detector probes (5 nM each) and Capture probe GenCp3 (60 nM) above with and without the Blocker probes (50 nM each).

TABLE 6.2

Results of Example 6

All Dt

All Dt

SCCmec +

All Dt

SCCmec −

Blocker

SCCmec +

Species
ADx ID
Type
Blocker
OD 492
B4A/B4B
OD 492
Blocker B5
OD 492

Staphylococcus aureus

#294
SCCmec type I
Positive
2.307
Positive
2.346
Positive
2.026

Staphylococcus aureus

#295
SCCmec type Ia
Positive
2.431
Positive
2.399
Positive
2.366

Staphylococcus aureus

#298
SCCmec type II
Positive
2.602
Positive
2.638
Positive
2.499

Staphylococcus aureus

#303
SCCmec type III
Positive
1.154
Positive
0.928
Positive
0.844

Staphylococcus aureus

#301
SCCmec type IIIa
Positive
1.417
Positive
1.255
Positive
1.060

Staphylococcus aureus

#304
SCCmec type IV
Positive
2.607
Positive
2.459
Positive
2.537

Staphylococcus aureus

#307
SCCmec type IV + PVL
Positive
2.167
Positive
2.523
Positive
2.503

Staphylococcus aureus

#460
Type V
Positive
1.161
Positive
0.998
Positive
0.755

Staphylococcus warneri

#351
MR-CNS
Positive
1.377
Negative
0.165
Negative
0.161

Staphylococcus

#352
MR-CNS
Negative
0.129
Negative
0.101
Negative
0.124

epidermidis

Staphylococcus hominis

#353
MR-CNS
Negative
0.113
Negative
0.112
Negative
0.146

Staphylococcus aureus

#156
MSSA
Negative
0.313
Negative
0.263
Negative
0.207

Results

Results of Example 6 are summarized in Table 6.2, which displays the absorbance values obtained at 492 nm and the positive/negative results for the samples. The cut off value is set at OD_{492 or 490}=0.500. Signals greater than or equal to the cut-off value are considered positive. Signals less than cut-off value are considered negative. Using the Capture (GenCp3) and all the SCCmec Detector Probes, all methicillin-resistant S. aureus strains (all SCCmec types) were positive as well as methicillin-resistant S. warneri.

By using Blocker probes designed to hybridize to chromosomal DNA of S. warneri (not S. aureus), specific detection of methicillin-resistant S. aureus was obtained.

The Blocker probes thus effectively prevent the Capture probe from hybridizing to chromosomal DNA of S. warneri, thereby allowing for specific detection of methicillin-resistant S. aureus.

Example 7
Use of Blocker Probes for Specific Detection of MRSA Using a Consensus Capture Probe

Table 7.1 displays the nucleobase sequences of the Capture (GenCp4) and Detector probe (Dt) and Blocker probes (B1A and BIB) used in the experiment below. The Capture probe is designed to hybridize to chromosomal DNA of S. aureus and S. epidermidis. The Detector probe is designed to hybridize to SCCmec type I, II & IV (not III and V) and the Blocker probes are designed to hybridize to chromosomal DNA of S. epidermidis (not S. aureus).

TABLE 7.1

Nucleobase Sequences

Probe

Type
Name
Sequence (5′ to 3′)

Capture
GenCP4
CAAATACAAAGTCGCTTTGCCCTTGTGTCA

(SEQ ID NO: 17)

Detector
Dt1
GCTATTATTTACTTGAAATGAAAGACTGCGGAGGCT

AACT

(SEQ ID NO: 2)

Blocker
B1A
TTTGACCTTGTGTCATGCGTGTTTGTAGTTCTTTAG

CGAG

(SEQ ID NO: 18)

Blocker
B1B
TGTAAACCATTGGAGCCACCTATGACAAATGTAAAG

TCGC

(SEQ ID NO: 19)

Method

Samples were prepared and assayed essentially according to the EVIGENE™ procedure using the Detector (5 nM) and Capture probes (60 nM) above with and without the Blocker probes (50 nM each).

Results

Results of Example 7 are summarized in Table 7.2, which displays the absorbance values obtained at 492 nm and the positive/negative results for the samples. Absorbance readings (OD₄₉₂>0.5) above background levels indicate the presence of the nucleic acid target in the sample.

TABLE 7.2

Results of Example 7

Dt1 (Type

Dt1 (Type

I, II, IV) +

AdvanDx

I, II, IV) −

Blocker

Species
ID
Type
Blocker
OD 492
B1A/B1B
OD 492

Staphylococcus

#9
SCCmec type I
Positive
1.965
Positive
1.796

aureus

Staphylococcus

#10
SCCmec type Ia
Positive
1.884
Positive
2.201

aureus

Staphylococcus

#11
SCCmec type II
Positive
1.854
Positive
1.800

aureus

Staphylococcus

#12
SCCmec type III
Negative
0.114
Negative
0.123

aureus

Staphylococcus

#13
SCCmec type IIIa
Negative
0.098
Negative
0.107

aureus

Staphylococcus

#14
SCCmec type IV
Positive
2.371
Positive
1.839

aureus

Staphylococcus

#460
Type V
Negative
0.114
Negative
0.100

aureus

Staphylococcus

#355
MR-CNS
Positive
1.310
Negative
0.114

epidermidis

Staphylococcus

#156
MSSA
Negative
0.143
Negative
0.124

aureus

The methicillin-resistant S. epidermidis and methicillin-resistant S. aureus strains of SCCmec type I, II or IV were all positive (OD₄₉₂>0.5) using the Capture and Detector Probe, whereas only the methicillin-resistant S. aureus strains were positive when using Blocker probes. The Blocker probes thus effectively prevent the Capture probe from hybridizing to chromosomal DNA of S. epidermidis, thereby allowing for specific detection of methicillin-resistant S. aureus without using a S. aureus-specific capture probe. This aspect of the invention provides an advantage over previous design schemes as the location of the probe is less limited.

Example 8
MRSA Detection with a Consensus Capture Probe, Blocker Probes and 9-mer LNA Detector Probe

Table 8.1 displays the nucleobase sequences of the Capture (GenCp4) and the Blocker probes (B1A and BIB) from Example 7, combined with the Detector probe (Dt5) from Example 4.

TABLE 8.1

Nucleobase Sequences

Probe

Type
Name
Sequence (5′ to 3′)

Capture
GenCP4
CAAATACAAAGTCGCTTTGCCCTTGTGTC

A

(SEQ ID NO: 17)

Detector
T1, 2, 4-Dt5
GaggctaaC

(SEQ ID NO: 11)

Blocker
B1A
TTTGACCTTGTGTCATGCGTGTTTGTAGT

TCTTTAGCGAG

(SEQ ID NO: 18)

Blocker
B1B
TGTAAACCATTGGAGCCACCTATGACAAA

TGTAAAGTCGC

(SEQ ID NO: 19)

LNA monomers are lowercase a, g, c, t, and DNA monomers are uppercase A, G, C, T

Method

Samples were prepared and assayed essentially according to the EVIGENE™ procedure using the Detector (Dt5, 50 nM) and Capture probe (GenCp4, 60 nM) above with and without the Blocker probes (B1A and BIB, 500 nM each).

Results

TABLE 8.2

Results of Example 8

Dt5 (Type

Dt5 (Type

I, II, IV) +

AdvanDx

I, II, IV) −

Blocker

Species
ID
Type
Blocker
OD 492
B1A/B1B
OD 492

Staphylococcus

#9
SCCmec type I
Positive
1.030
Positive
0.771

aureus

Staphylococcus

#10
SCCmec type Ia
Positive
1.168
Positive
0.967

aureus

Staphylococcus

#11
SCCmec type II
Positive
1.206
Positive
0.955

aureus

Staphylococcus

#12
SCCmec type
Negative
0.130
Negative
0.157

aureus

III

Staphylococcus

#13
SCCmec type
Negative
0.112
Negative
0.124

aureus

IIIa

Staphylococcus

#14
SCCmec type
Positive
1.106
Positive
1.130

aureus

IV

Staphylococcus

#460
Type V
Negative
0.122
Negative
0.129

aureus

Staphylococcus

#355
MR-CNS
Positive
0.561
Negative
0.155

epidermidis

Staphylococcus

#156
MSSA
Negative
0.124
Negative
0.223

aureus

Results of Example 8 are summarized in Table 8.2, which displays the absorbance values obtained at 492 nm and the positive/negative results for the samples. Absorbance readings (OD₄₉₂>0.5) above background levels indicate the presence of the nucleic acid target in the sample. This example shows that the Blocker probes prevent the Capture probe from hybridizing to chromosomal DNA of S. epidermidis, thereby allowing for specific detection of methicillin-resistant S. aureus without using a S. aureus-specific capture probe. The results also showed that mecA-positive S. aureus strains of SCCmec type I, II or IV tested positive with a 9-mer Detector probe hybridized together with the S. epidermidis Blocker probes.

Example 9
Detection of SCCmec Types I, II & IV Using 9-mer LNA/DNA or 9-mer LNA Probe

Table 9.1 displays the nucleobase sequences of the Capture probe GenCp3 with the Detector probe Dt5 (from Examples 4 and 8), Dt12 and Dt1.

TABLE 9.1

Nucleobase Sequences

Probe

Type
Name
Sequence (5′ to 3′)

Capture
GenCP3
TGTTCAATTAACACAACCCGCATCATTTG

ATGTGGGAATG

(SEQ ID NO: 5)

Detector
Dt1
GCTATTATTTACTTGAAATGAAAGACTGC

GGAGGCTAACT

(SEQ ID NO: 2)

Detector
T1, 2, 4-Dt5
GaggctaaC

(SEQ ID NO: 11)

Detector
T1, 2, 4-Dt12
gaggctaac

(SEQ ID NO: 20)

LNA monomers are lowercase a, g, c, t, and DNA monomers are uppercase A, G, C, T

Method

Samples were prepared and assayed essentially according to the EVIGENE™ procedure using the Detector probe Dt1(5 nM), 9-mer Dt5 LNA (50 nM) or 9-mer LNA (50 nM) with Capture probe GenCp3 (60 nM) above (Table 9.1).

Results

Results of Example 9 are summarized in Table 9.2, which displays the absorbance values obtained at 492 nm and the positive/negative results for the samples. The experiment showed that the methicillin-resistant S. aureus strains of SCCmec type I, II or IV were positive (OD₄₉₂>0.5) using the Capture GenCp3 with a 40-mer DNA or 9-mer mixed LNA/DNA Dt5 or a 9-mer only LNA (Dt12) Detector Probe.

The data also show that replacing some or all of the naturally occurring DNA nucleotides with non-naturally-occurring LNA monomers allows for use of short probes.

TABLE 9.2

Results of Example 9

Dt5 (Type

Dt12 (Type

Dt1 (Type

Species

OD 492
I, II, IV)
OD 492
I, II, IV)
OD 492
I, II, IV)
OD 492

Staphylococcus aureus

#295
SCCmec type Ia
Positive
1.547
Positive
1.832
Positive
2.218

Staphylococcus aureus

#298
SCCmec type II
Positive
1.616
Positive
1.944
Positive
2.075

Staphylococcus aureus

#16
SCCmec type IV
Positive
1.644
Positive
1.906
Positive
1.783

Staphylococcus aureus

#156
MSSA
Negative
0.144
Negative
0.168
Negative
0.200

Example 10
Detection of SCCmec Type III Detection with 9-mer LNA/DNA Probe

Table 10.1 displays the nucleobase sequences of the Capture probe (GenCp3) and the Detector probes Type III and Type III-Dt2.

TABLE 10.1

Nucleobase Sequences

Probe

Type
Name
Sequence (5′ to 3′)

Capture
GenCP3
TGTTCAATTAACACAACCCGCATCATTTG

ATGTGGGAATG

(SEQ ID NO: 5)

Detector
Type III
TAATCCAATATTTCATATATGTAATTCCT

CCACATCTCAT

(SEQ ID NO: 3)

Detector
Type III-Dt2
TtcctccaC

(SEQ ID NO: 21)

LNA monomers are lowercase a, g, c, t, and DNA monomers are uppercase A, G, C, T

Method

Samples were prepared and assayed essentially according to the EVIGENE™ procedure using the Detector probe Type III (5 nM) and 9-mer Type III-Dt2 LNA/DNA probe (50 nM) and Capture probe GenCp3 (60 nM) above (Table 10.1).

TABLE 10.2

Results of Example 10

Species
ADx ID
Type
Type III-Dt2
OD 492
Type III
OD 492

Staphylococcus

#303
SCCmec type
Positive
1.732
Positive
1.693

aureus

III

Staphylococcus

#301
SCCmec type
Positive
1.676
Positive
1.704

aureus

IIIa

Staphylococcus

#352
MR-CNS
Negative
0.214
Negative
0.117

epidermidis

Staphylococcus

#156
MSSA
Negative
0.273
Negative
0.158

aureus

Results of Example 10 are summarized in Table 10.2, which displays the absorbance values obtained at 492 nm and the positive/negative results for the samples. The experiment showed that the methicillin-resistant S. aureus strains of SCCmec type III were positive (OD₄₉₂>0.5) using Capture probe GenCp3 and a 40-mer DNA or 9-mer mixed LNA/DNA Detector Probe.

Example 11
Detection of SCCmec Type V Using 9-mer LNA Probe

Table 11.1 displays the nucleobase sequences of the Capture (GenCp3) with the Detector probes Type V and Type V-Dt4.

TABLE 11.1

Nucleobase Sequences

Probe

Type
Name
Sequence (5′ to 3′)

Capture
GenCP3
TGTTCAATTAACACAACCCGCATCATTTGATGTG

GGAATG

(SEQ ID NO: 5)

Detector
Type V
ACTTTAGTCAAATCATCTTCACTAGTGTAATTAT

CGAATG

(SEQ ID NO: 4)

Detector
Type V-
accattcac

Dt4

(SEQ ID NO: 22)

LNA monomers are lowercase a, g, c, t, and DNA monomers are uppercase A, G, C, T

Method

Samples were prepared and assayed essentially according to the EVIGENE™ procedure using the Detector probe Type V (5 nM) and 9-mer Type V-Dt4 LNA probe (50 nM) and Capture probe GenCp3 (60 nM) above (Table 10.1).

TABLE 11.2

Results of Example 11

Type

Species
Type
V-Dt4
OD 492
Type V
OD 492

Staphylococcus

SCCmec
Positive
0.647
Positive
0.588

aureus

type V

Staphylococcus

MSSA
Negative
0.155
Negative
0.113

aureus

Results of Example 11 are summarized in Table 11.2, which displays the absorbance values obtained at 492 nm and the positive/negative results for the samples. The experiment showed that the methicillin-resistant S. aureus strain of SCCmec type V was positive (OD₄₉₂>0.5) using the Capture GenCp3 and a 40-mer DNA or 9-mer LNA only Detector Probe. MSSA is methicillin sensitive Staphylococcus aureus.

Example 12
Use of Short LNA PCR Primers for Specific Detection of MRSA

Table 12.1 displays the nucleobase sequences of the PCR primers with specific LNA substitutions used in the PCR experiment below. The primers are designed to hybridize to genomic DNA of S. aureus or to a SCCmec type II as found in S. aureus strain Mu50.

TABLE 12.1

Nucleobase Sequences

Primer
Name
Sequence (5′ to 3′)

FORWARD

Dt1
F1
GCTATTATTTACTTGAAATGAAAGACTGCGGAGGCTA

ACT

(SEQ ID NO: 2)

Dt1
F5
GcGgAgGCTAACT

(SEQ ID NO: 23)

Dt1
F5-DNA
GCGGAGGCTAACT

(SEQ ID NO: 24)

Dt1
F7
AcTGcGGaGGCTAACT

(SEQ ID NO: 25)

Dt1
F8
cTGcGGaGGCTAAC

(SEQ ID NO: 26)

Dt1
F15
cgGaGGCTAAC

(SEQ ID NO: 27)

Dt1
F15-
CGGAGGCTAAC

DNA
(SEQ ID NO: 28)

Dt1
F16
cgGaGGCTAA

(SEQ ID NO: 29)

Dt1
F16-
CGGAGGCTAA

DNA
(SEQ ID NO: 30)

REVERSE

GenCP2
R1
CATTAGAAATACAAGGAAAGATGCTATCTTCCGAAGG

ATT

(SEQ ID NO: 31)

GenCP3
R1
CATTCCCACATCAAATGATGCGGGTTG

(SEQ ID NO: 32)

GenCP3
R3
ggGtTgTGTTAATT

(SEQ ID NO: 33)

GenCP3
R3-DNA
GGGTTGTGTTAATT

(SEQ ID NO: 34)

GenCP3
R7
ggGtTgTGTTAA

(SEQ ID NO: 35)

GenCP3
R7-DNA
GGGTTGTGTTAA

(SEQ ID NO: 36)

LNA monomers are lowercase a, g, c, t, and DNA monomers are uppercase A, G, C, T

Method

The DNA template was genomic bacterial DNA (Mu50, ATCC 700699), isolated with DNeasy Blood & Tissue kit (Qiagen). All DNA and LNA primers were suspended in 10 mM Tris. PCR amplification was performed in 50 μL reactions using 1 of each PCR primer, 800 ng or 8 ng genomic DNA and AccuPrime Taq DNA polymerase with AccuPrime buffer II, and 10 mM dNTPs (Invitrogen). Thermal cycling began with denaturation at 94.0° C. for 3 min followed by 30 cycles of 94.0° C. for 30 s, 55.0° C. for 30 s and 68.0° C. for 30 s. PCR product was visualized on 2% agarose gels with ethidium bromide and a 100 by DNA ladder (Invitrogen). Images of the gels were taken with a digital camera.

TABLE 12.2

Gel A
Gel B

Lane
Sample
Conc.
Lane
Sample
Conc.

2
100 bp DNA
0.5 μg
2
100 bp DNA ladder
0.5 μg

ladder

3
Cp2-R1 + Dt1-F5
Undil.
3
Cp3-R1 + Dt1-F5
Undil.

4
Cp2-R1 + Dt1-F5
1:100
4
Cp3-R1 + Dt1-F5
1:100

5
Cp2-R1 + Dt1-F7
Undil.
5
Cp3-R1 + Dt1-F7
Undil.

6
Cp2-R1 + Dt1-F7
1:100
6
Cp3-R1 + Dt1-F7
1:100

7
Cp2-R1 + Dt1-F8
Undil.
7
Cp3-R1 + Dt1-F8
Undil.

8
Cp2-R1 + Dt1-F8
1:100
8
Cp3-R1 + Dt1-F8
1:100

9
Cp2-R1 + Dt1-F1
Undil.
9
Cp3-R1 + Dt1-F1
Undil.

10
Cp2-R1 + Dt1-F1
1:100
10
Cp3-R1 + Dt1-F1
1:100

11
100 bp DNA
0.5 μg
11
100 bp DNA ladder
0.5 μg

ladder

Forward and reverse primer sequences are listed in Table 12.1. The forward Dt-F1 (SEQ ID NO:2) primer has the same sequence as Detector probe Dt1 used in the previous experiments. The primer pairs are listed in Table 12.2. Eight different primer pairs were tested for amplification of 800 ng (undiluted) or 8 ng (1:100) genomic DNA. All lanes of the agarose gels that were loaded with an aliquot of the completed amplification reaction showed a strong band of DNA of the expected length on 400 by (Gel A: forward primer: Dt-F1, -F5, -F7 or F8 paired with GenCp2-R1 reverse primer) and 200 by (Gel B: forward primer: Dt-F1, -F5, -F7 or F8 paired with GenCp3-R1 reverse primer).

TABLE 12.3

Lane
Sample

Band

Gel C

14-mer + 13-mer

1
100bp DNA ladder

2
Cp3-R3 + Dt1-F5
LNA-LNA
200 bp

3
Cp3-R3 + Dt1-F5-DNA
LNA-DNA
200 bp

4
Cp3-R3-DNA + Dt1-F5
DNA-LNA
None

5
Cp3-R3-DNA + Dt1-F5-DNA
DNA-DNA
None

12-mer + 13-mer

6
Cp3-R7 + Dt1-F5-DNA

200 bp

7
Cp3-R7-DNA + Dt1-F5
LNA-DNA
200 bp

8
Cp3-R7-DNA + Dt1-F5-DNA
DNA-LNA
None

9
Cp3-R7-DNA + Dt1-F5-DNA
DNA-DNA
None

10
100 bp DNA ladder

Gel D

14-mer + 11-mer

4
100bp DNA ladder

5
Cp3-R3 + Dt1-F15
LNA-LNA
200 bp

6
Cp3-R3 + Dt1-F15-DNA
LNA-DNA
Faint 200 bp

7
Cp3-R3-DNA + Dt1-F15
DNA-LNA
None

8
Cp3-R3-DNA + Dt1-F15-
DNA-DNA
None

DNA

9
100 bp DNA ladder

Gel E

14-mer + 10-mer

1
100bp DNA ladder

2
Cp3-R3 + Dt1-F16
LNA-LNA
Faint 200 bp

3
Cp3-R3 + Dt1-F16-DNA
LNA-DNA
None

4
Cp3-R3-DNA + Dt1-F16
DNA-LNA
None

5
Cp3-R3-DNA + Dt1-F16-
DNA-DNA
None

DNA

6
100 bp DNA ladder

Forward and reverse primer sequences are listed in Table 12.1. The primer pairs are listed in Table 12.3. In Gel C, eight different primer pairs were tested for amplification of 800 ng (undiluted) DNA. All amplicons (200 bp) were made with a forward Dt1-F5 (13-mer LNA/DNA primer) or Dt-F5-DNA (13-mer DNA primer), paired with GenCP3-R3 (14-mer LNA/DNA primer) or GenCP3-R7 (12-mer LNA/DNA) as reverse primers. In Gel D, four different primer pairs were tested for amplification of 800 ng (undiluted) DNA (Gel D, Table 12.3). All amplicons (200 bp) were made with a forward Dt1-F15 (11-mer LNA/DNA) primer or Dt-F11-DNA (11-mer DNA) primer, and GenCP3-R3 (14-mer LNA/DNA), reverse primer. In Gel E, four different primer pairs were tested for amplification of 800 ng (undiluted) DNA (Gel D, Table 12.3). All amplicons (200 bp) were made with a forward Dt1-F16 (10-mer LNA/DNA) primer or Dt-F16-DNA (10-mer DNA) primer, and GenCP3-R3 (14-mer LNA/DNA) reverse primer.

In Gel C, lanes 2 and 3 had strong bands of DNA of the expected length as a product of the amplification reaction. Lanes 6 and 7 had fainter bands. Other primer combinations did not result in detectable product.

In Gel D, lane 2 had strong band and lane 3 had faint band of DNA of the expected length of amplification product. See Table 12.3 for the primer combinations. Other primer combinations did not result in detectable product.

In Gel E, lanes 2 had faint band of DNA of the expected length of amplification product. See Table 12.3 for the primer combinations. Other primer combinations did not result in detectable product.

The PCR experiments showed that LNA incorporation improves the efficiency of short, low-T_m, (AT-rich) primers. Three or four LNA subunits were incorporated into each primer, because this was sufficient to elevate the predicted melting temperatures into the range that resulted in products visible on stained agarose gels as good bands. Previous work showed that PCR failed when primers had more than three LNAs (Ugozzoli, L. A. et al., Anal. Biochem 324:143-152, 2004) but the results on Gels B, C, D and E demonstrated that a 14-mer, or 12-mer primer containing 4 LNA monomers successfully primed amplification.

The experiment showed that the methicillin-resistant S. aureus strain Mu50 of SCCmec type II was detected by PCR by using a 13-, 11- or a 10-mer forward LNA/DNA and a 14- or a 12-mer reverse LNA/DNA primer, but not detected by PCR when using DNA-only primers with the same sequence and length.

	Number	Date	Country
Parent	PCT/US08/06285	May 2008	US
Child	12620187		US

DETECTION OF METHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS

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