Patent Grant
7972777
This application is the national phase under 35 U.S.C. §371 of PCT International Application No. PCT/IE00/00066 which has an International filing date of May 15, 2000, which designated the United States of America and was published in English.
This invention relates to the identification of target sequences for use in nucleic acid assays for the detection and identification of prokaryotic and/or eukaryotic organisms.
The ssrA gene, which encodes a small stable high copy number RNA transcript (tmRNA), is found in all bacteria and has recently been identified in chloroplasts and diatoms. It has a dual function both as a tRNA and as an mRNA molecule and is involved in rescuing truncated mRNAs which have lost stop codons, facilitating trans-translation of truncated peptides prior to protease degradation (Keiler, K. C. et al. (1996), Science, 271, 990-993). The unique function of tmRNAs has directed researchers to analyse the relationship of the secondary structure of these molecules with their function. These studies have focussed on the conservation of the secondary structure of tmRNAs from different microorganisms, and on the evolutionary significance and functional relevance of such structural conservation. Studies were carried out by Matveeva, O et al (1998), Vol. 16, No. 13, 1374-1375 to investigate oligonucleotide binding to RNA molecules using tmRNA as a model of RNA containing secondary structure. The studies did not have as their objective the identification of sites in tmRNA with the goal of designing antisense oligonucleotide for therapeutic purposes.
The number of nucleic acid targets/probes for bacterial diagnostics is currently limited. As such, the need to identify and characterise novel DNA and RNA targets for diagnostic purposes is now seen as a priority. Target nucleic acid sequences for the development of probes can be for example, plasmids, ribosomal RNA genes, intergenic regions, genes encoding virulence factors or random genomic DNA fragments. In addition, a number of RNA molecules have been described which are used as targets for RNA-based detection for example, ribosomal RNA and RNase P.
The basis of any nucleic acid-based probe assay is the requirement for well characterised nucleic acid sequences which are present in all prokaryotes and eukaryotes under study. For reliable detection of a prokaryotic or eukaryotic organism, the nucleic acid probes used should be highly specific (i.e. not cross-react with nucleic acids from other organisms) and highly sensitive (i.e. most or all strains of the organism to be detected should react with the probe). Therefore, preferred target sequences would be present in all strains of the organism concerned. Such sequences would have significant sequence variability to allow differentiation of the species concerned from other closely related species but, on the other hand, have sufficient sequence conservation to allow the detection of all strains of the species concerned. In general, the precise identification of a nucleic acid sequence, which could form the basis of a specific nucleic acid probe assay, is tedious, difficult and uncertain. To date there are few general approaches which would facilitate the development of nucleic acid probes for a wide variety of microorganisms. The nucleic acid sequences which have been identified as potentially useful targets for probe development are, for example, rRNA genes and RNA, and the rRNA 16S/23S intergenic region.
The majority of nucleic acid probe/target assays centre on the high copy number ribosomal RNAs (rRNA) and rRNA 16S/23S spacer regions (European Patent No. 0 395 292) of the bacterial cell for the purposes of detection and identification. A number of successful commercial bacterial diagnostic kits have been marketed based on these rRNA probes/targets for the detection of a variety of microrganisms. These include a range of commercial probe kits based on the 16S rRNA gene marketed by Gen-probe Inc. San Diego Calif., and DNA probes based on the 16S/23S spacer region marketed by Innogenetics N.V. Ghent, Belgium. However, many of these diagnostic kits have limitations, including lack of sensitivity due to low copy-number target sequences and lack of specificity due to sequence identity between closely related organisms in many cases.
Nucleic acid-based methods that could be applied directly to samples to give an indication of the viability of any microbes present therein would be of enormous significance for food, industrial, environmental and medical applications.
A disadvantage of DNA-based methods is that they do not distinguish between living and dead organisms. Some studies have focussed on using rRNA and mRNA as indicators of cell viability (Sheridan, G. E. C. et al. (1998) Applied and Environmental Microbiology, 64, 1313-1318). However, these sequences are not satisfactory targets as rRNA and mRNA can be present in bacterial cells up to 48 hours after cell death.
With the advent of nucleic acid based microarray-like formatting, incorporating simultaneous monitoring of multiple nucleic acid targets, there is now a clear requirement to identify and characterise novel nucleic acid sequences for use as probes and/or target regions to detect and identify viable prokaryotic and eukaryotic cells.
The invention provides use of the ssrA gene or a fragment thereof as a target region in a nucleic acid probe assay for a prokaryotic or eukaryotic organism.
Thus, the invention has application in relation to all organisms other than viruses.
No other nucleic acid probe assay has been reported which uses regions of the ssrA gene as a target region to detect and identify species of prokaryotes and eukaryotes with the attendant advantages.
According to one embodiment of the invention a fragment of the ssrA gene molecule corresponding to a region of high homology from the 5′ end of the DNA molecule can be used as a universal target region.
In an alternative embodiment of the invention a fragment of the ssrA gene molecule corresponding to a region of high homology from the 3′ end of the DNA molecule can be used as a universal target region.
In a further embodiment of the invention a fragment of the ssrA gene molecule corresponding to a region of low homology can be used as a target region in a nucleic acid probe assay to distinguish between species.
In a still further embodiment of the invention a fragment of the ssrA gene molecule corresponding to a region of low homology can be used as a target region for the generation of a genus specific probe.
As hereinafter described nucleotide sequence alignments of ssrA gene sequences from different organisms show that the 5′ and 3′ regions of these molecules demonstrate a high degree of homology and are therefore useful as universal target regions. The ssrA genes also demonstrate a more significant degree of nucleotide sequence variability between closely related organisms than any other bacterial high copy number RNA. These variable regions are ideal targets for nucleic acid assays to distinguish between species.
The invention also provides use of tmRNA, an RNA transcript of the ssrA gene, or a fragment thereof as a target region in a nucleic acid probe assay for a prokaryotic or eukaryotic organism.
According to one embodiment of this aspect of the invention a fragment of a tmRNA molecule corresponding to a region of high homology from the 5′ end of the tmRNA molecule can be used as a universal target region.
Alternatively, a fragment of a tmRNA molecule corresponding to a region of high homology from the 3′ end of the tmRNA molecule can be used as a universal target region.
According to a further embodiment of this aspect of the invention a fragment of a tmRNA molecule corresponding to a region of low homology can be used as a target region in a nucleic acid probe assay to distinguish between species.
According to a still further embodiment a fragment of a tmRNA molecule corresponding to a region of low homology can be used as a target region for the generation of a genus specific probe.
The nucleic acid probe (DNA or RNA) in accordance with the invention typically consists of at least 10 nucleotides of the ssrA gene and/or tmRNA transcript or their complementary sequence and is used in a nucleic acid probe hybridisation assay for a prokaryotic or eukaryotic organism. Probe hybridisation to its complementary sequence is typically revealed by labelling the nucleic acid probe with a radioactive or non-radioactive (e.g. colorimetric or fluorimetric) label.
In preferred embodiments said ssrA gene fragment or said tmRNA fragment can be used as the basis of a primer to be used in an amplification procedure.
Universal oligonucleotide primers directed to the 5′ and 3′ regions of either the ssrA gene or the tmRNA sequence can be used in accordance with the invention to amplify the ssrA gene or its encoding tmRNA from a wide variety of bacteria, facilitating amplification of a wide range of organisms simultaneously, whilst also enabling specific nucleic acid probe hybridisation and detection.
Preferably, the product of the amplification procedure is used as a target region in a nucleic probe assay.
Further, preferably, a cDNA transcript of a tmRNA molecule is used as a probe in a nucleic acid hybridisation assay.
Such assays can be carried out in vitro or in situ.
The target region as defined herein can be used as the basis of an assay for distinguishing between living and dead prokaryotic or eukaryotic organisms.
In contrast to rRNA and mRNA which can be present in bacterial cells following cell death, tmRNA is rapidly degraded in dead organisms. Thus, tmRNA can be a useful target for distinguishing between living and dead prokaryotic or eukaryotic organisms either directly by nucleic acid probe hybridisation to isolated bacterial RNA, or by combined RNA amplification and nucleic acid probe hybridisation to the amplified product.
Preferably, the target region is used in a multiple probe format for broad scale detection and/or identification of prokaryotic or eukaryotic organisms.
An ssrA gene probe or a tmRNA transcript probe in accordance with the invention can be linked to a microarray gene chip system for the broad scale high throughput detection and identification of prokaryotic or eukaryotic organisms.
A target region in accordance with the invention can also be used as a probe in an assay to detect prokaryotic or eukaryotic organisms in a sample of matter.
Such a sample of matter can include biological samples such as samples of tissue from the respiratory tract, the uro-genital tract or the gastrointestinal tract, or body fluids such as blood and blood fractions, sputum or cerebrospinal fluid.
An assay in accordance with the invention can also be carried out on food samples, environmental samples including air, water, marine and soil samples, and plant and animal derived samples.
According to the invention a fragment of the ssrA gene or the tmRNA transcript can also be used in an assay to obtain a DNA profile of a prokaryotic or eukaryotic organism and, thereby, distinguish between strains of the same species.
Nucleic acid sequence alignments have shown that sequence variation occurs in the ssrA gene and the tmRNA transcript within individual species. This intra-species sequence variation can be used to distinguish between strains of the same species for epidemiology, tracing of infectious agents for example, in outbreaks, or for population studies.
Other applications of the invention include the use of the ssrA gene, the tmRNA transcript or a DNA sequence complementary thereto, or a fragment thereof, to design an agent directed against infectious prokaryotic or eukaryotic organisms for therapeutic purposes.
Such agents can include antisense mRNA or oligonucleotides, ribozymes, and antagonistic peptides and are suitable for use in any kind of medical condition.
Thus, the invention can be used for the detection of viable organisms only in biological samples using the tmRNA target. Thus, during and following any anti-infectious agent drug treatment, the tmRNA target can be used to monitor the efficacy of the therapy on those specific infectious agents (e.g. antimicrobial and/or anti-parasitic treatments).
In one embodiment, the target region is used to monitor the efficacy of drug therapies against infectious agents.
In another embodiment, the target region is used to monitor the viability and level of health-promoting organisms in the gastrointestinal tract.
This aspect of the invention relates, for example, to the introduction into the gut flora of health-promoting (probiotic) organisms contained in for example yoghurt or other food to improve health. There is an interest and need to continuously monitor the presence and levels of these organisms to ensure their continued function in promoting health. The tmRNA region can be used as a target to detect viable organisms, for example in faeces, so as to monitor the presence of the health promoting organisms.
In a further embodiment, the assay is used for the quantification of prokaryotic or eukaryotic organisms.
When using probe hybridisation and/or in vitro amplification to detect organisms in a sample it is possible to determine the number of organisms present, based on the signal intensity. Real-time methods of in vitro amplification can also be used to enable the quantification of organisms in a sample. Thus, the ability to quantify the number of organisms in a sample can be important in clinical situations for treatment purposes, for example for antibiotic or other treatments or for monitoring treatment efficacy.
A still further application of the invention is the use of a database of ssrA gene sequences to identify a prokaryotic or eukaryotic organism.
The invention provides a variety of probes for the 5′ and 3′ homologous regions and the variable regions of the ssrA gene and tmRNA sequences, the probes being derived from these sequences or sequences complementary thereto. Representative sequences are as follows:
Actinobacillus actinomycetemcomitans ssrA
Actinobacillus actinomycetemcomitans tmRNA
Aeromonas salmonicida ssrA, Internal Partial
Aeromonas salmonicida tmRNA, Internal Partial
Alcaligenes eutrophus ssrA
Alcaligenes eutrophus tmRNA
Aquifex aeolicus ssrA
Aquifex aeolicus tmRNA
Bacillus megaterium ssrA, Internal Partial
Bacillus megaterium tmRNA, Internal Partial
Bacillus subtilis ssrA
Bacillus subtilis tmRNA
Bordetella pertussis ssrA
Bordetella pertussis tmRNA
Borrelia burgdorferi ssrA
Borrelia burgdorferi tmRNA
Campylobacter jejuni ssrA
Campylobacter jejuni tmRNA
Chlamydia trachomatis (D/UW-3/CX) ssrA
Chlamydia trachomatis (D/UW-3/CX) tmRNA
Chlamydia trachomatis (Mouse Pneumonitis) ssrA
Chlamydia trachomatis (Mouse Pneumonitis) tmRNA
Chlorobium tepidum ssrA
Chlorobium tepidum tmRNA
Cyanophora paradoxa (Alga) Cyanelle ssrA
Cyanophora paradoxa (Alga) Cyanelle tmRNA
Clostridium acetobutylicum ssrA, 3′ Partial
Clostridium acetobutylicum tmRNA, 3′ Partial
Deinococcus radiodurans ssrA
Deinococcus radiodurans tmRNA
Desulfovibrio desulfuricans ssrA, Internal Partial
Desulfovibrio desulfuricans tmRNA, Internal Partial
Dichelobacter nodosus ssrA, 3 Partial
Dichelobacter nodosus tmRNA, 3 Partial
Enterococcus faecalis ssrA
Enterococcus faecalis tmRNA
Escherichia coli ssrA
Escherichia coli tmRNA
Haemophilus influenzae ssrA
Haemophilus influenzae tmRNA
Helicobacter pylori (ATCC 43504) ssrA, Internal Partial
Helicobacter pylori (ATCC 43504) tmRNA, Internal Partial
Helicobacter pylori (strain 26695) ssrA
Helicobacter pylori (Strain 26695) tmRNA
Klebsiella aerogenes (NCTC 9528) ssrA, Internal Partial
Klebsiella aerogenes (NCTC 9528) tmRNA, Internal Partial
Lactobacillus lactis (NCTC 662) ssrA, Internal Partial
Lactobacillus lactis (NCTC 662) tmRNA, Internal Partial
Legionella pneumophila ssrA, Internal Partial
Legionella pneumophila tmRNA, Internal Partial
Listeria grayi ssrA, Internal Partial
Listeria grayi tmRNA, Internal Partial
Listeria innocua ssrA, Internal Partial
Listeria innocua tmRNA, Internal Partial
Listeria monocytogenes (NCTC 7973) ssrA, Internal Partial
Listeria monocytogenes (NCTC 7973) tmRNA, Internal Partial
Listeria monocytogenes (NCTC 11994) ssrA, Internal Partial
Listeria monocytogenes (NCTC 11994) tmRNA, Internal Partial
Listeria murrayi ssrA, Internal Partial
Listeria murrayi tmRNA, Internal Partial
Listeria welshimeri ssrA, Internal Partial
Listeria welshimeri tmRNA, Internal Partial
Marinobacter hydrocarbonoclasticus ssrA, Internal Partial
Marinobacter hydrocarbonoclasticus tmRNA, Internal Partial
Mycobacterium avium ssrA, Internal Partial
Mycobacterium avium tmRNA, Internal Partial
Mycobacterium bovis ssrA, Internal Partial
Mycobacterium bovis tmRNA, Internal Partial
Mycobacterium leprae ssrA
Mycobacterium leprae tmRNA
Mycobacterium paratuberculosis ssrA, Internal Partial
Mycobacterium paratuberculosis tmRNA, Internal Partial
Mycobacterium tuberculosis ssrA
Mycobacterium tuberculosis tmRNA
Mycoplasma capricolumn ssrA
Mycoplasma capricolumn tmRNA
Mycoplasma genitalium (ATTC 33530, #1) ssrA
Mycoplasma genitalium (ATTC 33530, #1) tmRNA
Mycoplasma genitalium (ATTC 33530, ≯2) tmRNA, Internal Partial
Mycoplasma genitalium (ATTC 33530, #2) tmRNA, Internal Partial
Mycoplasma Pneumophila ssrA
Mycoplasma pneumophila tmRNA
Neisseria gonorrhoeae (ATCC 19424) ssrA, Internal Partial
Neisseria gonorrhoeae (ATCC 19424) tmRNA, Internal Partial
Neisseria gonorrhoeae (FA 1090) ssrA
Neisseria gonorrhoeae (FA 1090) tmRNA
Neisseria meningitidis ssrA
Neisseria meningitidis tmRNA
Nostoc muscorum PCC7120 ssrA
Nostoc muscorum PCC7120 tmRNA
Odontella sinensis (diatom) Chloroplast ssrA
Odontella sinensis (Diatom) Chloroplast tmRNA
Porphyra purpureum (Red Alga) Chloroplast ssrA
Porphyra purpureum (Red Alga) Chloroplast tmRNA
Porphyromonas gingivalis ssrA
Porphyromonas gingivalis tmRNA
Proteus rettgeri ssrA (NCTC 10975), Internal Partial
Proteus rettgeri tmRNA (NCTC 10975), Internal Partial
Pseudoalteromonas haloplanktoni ssrA, Internal Partial
Pseudoalteromonas haloplanktoni tmRNA, Internal Partial
Pseudomonas aeruginosa ssrA
Pseudomonas aeruginosa tmRNA
Salmonella typhimurium ssrA
Salmonella typhimurium tmRNA
Shewanella putrefaciens ssrA
Shewanella putrefaciens tmRNA
Staphylococcus aureus ssrA
Staphylococcus aureus tmRNA
Streptococcus gordonii ssrA
Streptococcus gordonii tmRNA
Streptococcus mutans ssrA
Streptococcus mutans tmRNA
Streptococcus pneumoniae ssrA
Streptococcus pneumoniae tmRNA
Streptococcus pyogenes ssrA
Streptococcus pyogenes tmRNA
Synechococcus sp. PCC6301 ssrA
Synechococcus sp. PCC6301 tmRNA
Synechocystis sp. PCC6803 ssrA
Synechocystis sp. PCC6803 tmRNA
Thermotoga maritima ssrA
Thermotoga maritima tmRNA
Thermus thermophilus ssrA
Thermus thermophilus tmRNA
Treponema pallidum ssrA
Treponema pallidum tmRNA
Vibrio cholerae ssrA
Vibrio cholerae tmRNA
Yersinia pestis ssrA
Yersinia pestis tmRNA
Campylobacter fetus ssrA, Internal Partial
Campylobacter fetus tmRNA, Internal Partial
Campylobacter coli (BM2509) ssrA, Internal Partial
Campylobacter coli (BM2509) tmRNA, Internal Partial
Camplyobacter Chicken Isolate ssrA, Internal Partial
Camplyobacter Chicken Isolate tmRNA, Internal Partial
Clostridium perfringens ssrA, Internal Partial
Clostridium perfringens tmRNA, Internal Partial
Haemophilus ducreyi (NCTC 10945) ssrA, Internal Partial
Haemophilus ducreyi (NCTC 10945) tmRNA, Internal Partial
Listeria innocua (Food Isolate #1) ssrA, Internal Partial
Listeria innocua (Food Isolate #1) tmRNA, Internal Partial
Listeria innocua (Food Isolate #2) ssrA, Internal Partial
Listeria innocua (Food Isolate #2) tmRNA, Internal Partial
Listeria innocua (Food Isolate #3) ssrA, Internal Partial
Listeria innocua (Food Isolate #3) tmRNA, Internal Partial
Listeria innocua (ATCC 12210) ssrA, Internal Partial
Listeria innocua (ATCC 12210) tmRNA, Internal Partial
Listeria ivanovii (NCTC 11846) ssrA, Internal Partial
Listeria ivanovii (NCTC 11846) tmRNA, Internal Partial
Listeria seeligeri (NCTC 11856) ssrA, Internal Partial
Listeria seeligeri (NCTC 11856) tmRNA, Internal Partial
Salmonella enteritidis ssrA, Internal Partial
Salmonella enteritidis tmRNA, Internal Partial
Staphylococcus epidermidis (NCTC 11047) ssrA, Internal Partial
Staphylococcus epidermidis (NCTC 11047) tmRNA, Internal Partial
Streptococcus agalactiae (NCTC 8181) ssrA, Internal Partial
Streptococcus agalactiae (NCTC 8181) tmRNA, Internal Partial
Bordetella bronchiseptica ssrA
Bordetella bronchiseptica tmRNA
Chlamydia pneumoniae (CWL029), ssrA
Chlamydia pneumoniae (CWL029) tmRNA
Francisella tularensis ssrA
Francisella tularensis tmRNA
Guillardia theta (Plastid) ssrA
Guillardia theta (Plastid) tmRNA
Thalassiosira Weissflogii (Plastid) ssrA
Thalassiosira Weissflogii (Plastid) tmRNA
Helicobacter pylori ssrA, (Clinical Isolate 1), Internal Partial
Helicobacter pylori tmRNA, (Clinical Isolate 1), Internal Partial
Helicobacter pylori ssrA, (Clinical Isolate 2), Internal Partial
Helicobacter pylori tmRNA, (Clinical Isolate 2), Internal Partial
Listeria seeligeri (NCTC 11856) ssrA, Internal Partial
Listeria seeligeri (NCTC 11856) tmRNA, Internal Partial
Listeria ivanovii (NCTC 11846) ssrA, Internal Partial
Listeria ivanovii (NCTC 11846) tmRNA, Internal Partial
Mycobacterium africanum (Clinical Isolate) ssrA, Internal Partial
Mycobacterium africanum (Clinical Isolate) tmRNA, Internal Partial
Mycobacterium gordonae(Clinical Isolate) ssrA, Internal Partial
Mycobacterium gordonae(Clinical Isolate) tmRNA, Internal Partial
Mycobacterium kansasii (Clinical Isolate) ssrA, Internal Partial
Mycobacterium kansasii (Clinical Isolate) tmRNA, Internal Partial
Mycobacterium chelonae ssrA, Internal Partial
Mycobacterium chelonae tmRNA, Internal Partial
Mycobacterium szulgai (ATCC 35799) ssrA, Internal Partial
Mycobacterium szulgai (ATCC 35799) tmRNA, Internal Partial
Mycobacterium malmoense (Clinical Isolate) ssrA, Internal Partial
Mycobacterium malmoense (Clinical Isolate) tmRNA, Internal Partial
Mycobacterium flavescens ssrA, Internal Partial
Mycobacterium flavescens tmRNA, Internal Partial
Mycobacterium marinum ssrA, Internal Partial
Mycobacterium marinum tmRNA, Internal Partial
Mycobacterium microti (Environmental Isolate) ssrA, Internal Partial
Mycobacterium microti (Environmental Isolate) tmRNA, Internal Partial
Mycobacterium smegmatis (ATCC 10143) ssrA, Internal Partial
Mycobacterium smegmatis (ATCC 10143) ssrA, Internal Partial
Mycobacterium xenopi (Clinical Isolate) ssrA, Internal Partial
Mycobacterium xenopi (Clinical Isolate) tmRNA, Internal Partial
Mycobacterium intracellulare (NCTC 10425) ssrA, Internal Partial
Mycobacterium intracellulare (NCTC 10425) tmRNA, Internal Partial
Mycobacterium scrofulaceum (NCTC 10803) ssrA, Internal Partial
Mycobacterium scrofulaceum (NCTC 10803) tmRNA, Internal Partial
Nocardia asteroides ssrA, Internal Partial
Nocardia asteroides tmRNA, Internal Partial
Salmonella enteritidis ssrA, Internal Partial
Salmonella enteritidis tmRNA, Internal Partial
Staphylococcus epidermidis (NCTC 11047) ssrA, Internal Partial
Staphylococcus epidermidis (NCTC 11047) tmRNA, Internal Partial
Streptococcus agalactiae (NCTC 8181) ssrA, Internal Partial
Streptococcus agalactiae (NCTC 8181) tmRNA, Internal Partial
Of the above sequences SEQ ID NOs 47 to 62, 65 to 68, 71 and 72, and 99, 159 to 168 and 176-217 are novel sequences.
The above mentioned sequences can be used to form a database of ssrA gene sequences which can be used to identify a bacterial species, or for the generation of nucleic acid diagnostic assays.
Representative probes identified in accordance with the invention are as follows:
Salmonella:
1) Genius specific probe:
Mycobacteria:
2) Oligonucleotide probe for detection of tuberculosis complex
3) Oligonucleotide probes for detection of M. avium and M. paratuberculosis Sequences
Listeria:
4) Oligonucleotide probe used in the determination of tmRNA integrity after heat killing treatment of cells:
Escherichia coli:
5) Oligonucleotide probe used in the determination of tmRNA integrity after heat killing treatment of cells:
Further representative primers identified in accordance with the invention are as follows:
Mycobacteria:
1) Degenerative oligonucleotide primers for the amplification of all mycobacterial sequences
5′ Primer
3′ Primer
2) Oligonucleotide primers for the amplification of M. avium and M. paratuberculosis
In the accompanying drawings:
The invention will be further illustrated by the following Examples.
A comparative primary nucleotide sequence alignment of available tmRNA sequences using the Clustal W nucleic acid alignment programme demonstrated that tmRNA sequences from prokaryotes show a more significant degree of nucleotide sequence variability and non-homology than other bacterial high copy number RNA, as demonstrated in Table 1.
Bacillus
subtilus vs.
Escherichia
coli vs.
Mycobacterium
Vibrio
cholerae
tuberculosis
These regions of non-homology between tmRNA sequences from different bacteria are located in the middle of the molecule, and the extent of nucleotide sequence non-homology within the tmRNA molecule indicated that genus as well as species specific probes could be generated to distinguish between and/or detect bacteria.
Nucleotide sequence alignments had previously shown that the 5′ and 3′ flanking regions of the tmRNA molecules share a high degree of homology both within species and within genus. This observation indicated that universal oligonucleotide primers could be generated to amplify the ssrA gene or its encoding tmRNA from a wide variety of bacteria.
We have now demonstrated that these regions of homology and non-homology within the nucleotide sequence of tmRNA molecules from different organisms can be used as the basis of identifying and detecting organisms at the molecular level.
A nucleotide sequence alignment of the E. coli (SEQ ID NO. 37) and V. cholerae (SEQ ID NO. 127) ssrA sequences as depicted in
The sequence of the V. cholerae tmRNA specific probe is
Total RNA was isolated from liquid cultures of E. coli and V. cholerae at the mid-exponential phase and the stationary phase of growth. Equivalent amounts of the isolated total RNA were electrophoresed on a denaturing formaldehyde agarose gel and blotted onto HYBOND-N nylon membrane as shown in
Lane 1: Total E. coli RNA mid-log phase
Lane 2: Total V. cholerae RNA mid-log phase
Lane 3: Total E. coli RNA stationary phase
Lane 4: Total V. cholerae RNA stationary phase
The resulting Northern blot was then hybridised with the V. cholerae tmRNA specific probe end-labelled with γP32. The results of the hybridisation experiment shown in
Clustal W alignment of all available ssrA gene and tmRNA sequences indicated that degenerate oligonucleotide primers could be designed to amplify ssrA gene and tmRNA nucleotide sequences for a wide variety of organisms.
Degenerate oligonucleotide primers were synthesised to PCR amplify ssrA gene sequences from total genomic DNA preparations from a broad range of bacteria.
The sequences of the synthesised degenerate oligonucleotides are as follows:
The products of PCR reactions were electrophoresed on an agarose gel and a 350 base pair (approx.) PCR product was amplified in all cases, as shown in
In
Lane A: Molecular weight marker V
Lane 1: Escherichia coli
Lane 2: Salmonella poona
Lane 3: Klebsiella aerogenes
Lane 4: Proteus mirabilis
Lane 5: Proteus rettgeri
Lane 6: Aeromonas hydrophilia
Lane 7: Staphyloccus aureus
Lane 8: Enterococcus faecalis
Lane 9: Lactobacillus lactis
Lane 10: Bacillus subtilus
Lane 11: Listeria monocytogenes
Lane 12: Listeria innocua
Lane 13: Listeria murrayi
Lane 14: Listeria welshimeri
Lane 15: Listeria grayi
Lane 16: Mycobacterium bovis
Lane B: Molecular weight marker V
The universal primers amplified the ssrA gene from both Gram positive and Gram negative bacteria, as shown in Table 2.
Escherichia
coli
Salmonella
poona
Klebsiella
aerogenes
Proteus
mirabilis
Proteus
rettgeri
Aeromonas
hydrophilia
Staphyloccus
aureus
Enterococcus
faecalis
Lactobacillus
lactis
Bacillus
subtilus
Listeria
monocytogenes
Listeria
innocua
Listeria
murrayi
Listeria
welshimeri
Listeria
grayi
Mycobacterium
bovis
The PCR products amplified from genomic DNA from the Listeria species of bacteria and that from the M. bovis bacterium, from Example 2, were subcloned into a T-tailed plasmid vector for the purposes of DNA sequencing. Three recombinant clones were selected for each species and sequenced by the di-deoxy sequencing method. The sequence of both DNA strands for each subclone was determined.
The nucleotide sequence determined for the M. bovis ssrA gene shared 100% homology with the Mycobacterium tuberculosis ssrA gene sequence.
A clustal W alignment of the novel ssrA gene sequences obtained for the Listeria species (SEQ ID NOS 51, 53, 55, 59 and 61) is shown in
In
To further illustrate that the ssrA gene/tmRNA nucleic acid target is a suitable target for bacterial diagnostics, a comparative alignment of the L. monocytogenes ssrA gene nucleotide sequence (SEQ ID NO. 55) with the available B. subtilis ssrA gene nucleotide sequence (SEQ ID NO. 11) (a phylogenetically closely related bacteria to Listeria) was carried out as shown in
Using the Listeria genus ssrA gene/tmRNA nucleotide sequence alignment of Example 4, regions of the ssrA gene/tmRNA nucleotide sequence were analysed to determine their suitability for the generation of genus-specific amplification primers, and genus-specific and species-specific oligonucleotide probes. In this analysis, regions which demonstrated the greatest sequence differences to B. subtilis, were selected in the design of these amplification primers and probes.
The sequences of the synthesised oligonucleotides are as follows:
Listeria genus specific hybridisation probe
L.
monocytogenes species specific hybridisation
The genus specific amplification primers Ltm1 and Ltm2 were used in a series of PCR reactions with total genomic DNA from twenty different strains as the template in each case. Only ssrA gene sequences from the Listeria species were amplified (260 base pair product) with these primers (
In
Lane A: Molecular weight marker V
Lane 1: E. coli
Lane 2: S. poona
Lane 3: K. aerogenes
Lane 4: P. mirabilis
Lane 5: P. rettgeri
Lane 6: A. hydrophilia
Lane 7: S. aureus
Lane 8: E. faecalis
Lane 9: L. lactis
Lane 10: B. subtilus
Lane 11: L. monocytogenes strain 1
Lane 12: L. monocytogenes strain 2
Lane 13: L. monocytogenes strain 3
Lane 14: L. monocytogenes strain 4
Lane 15: L. monocytogenes clinical isolate
Lane 16: L. innocua
Lane 17: L. murrayi
Lane 18: L. welshimeri
Lane 19: L. grayi
Lane 20: M. bovis
Lane B: Molecular weight marker V
Escherichia
coli
Salmonella
poona
Klebsiella
aerogenes
Proteus
mirabilis
Proteus
rettgeri
Aeromonas
hydrophilia
Staphyloccus
aureus
Entrococcus
faecalis
Lactobacillus
lacus
Bacillus
subtilus
Listeria
monocytogenes strain 1
Listeria
monocytogenes strain 2
Listeria
monocytogenes strain 3
Listeria
monocytogenes strain 4
Listeria
monocytogenes clinical
Listeria
innocua
Listeria
murrayi
Listeria
welshimeri
Listeria
grayi
Mycobacterium
bovis
The Listeria genus specific oligonucleotide probe, LGtm, was hybridised to the Southern blot depicted in
In
Lane A: Molecular weight marker V
Lane 1: Escherichia coli
Lane 2: Salmonella poona
Lane 3: Klebsiella aerogenes
Lane 4: Proteus mirabilis
Lane 5: Proteus rettgeri
Lane 6: Aeromonas hydrophilia
Lane 7: Staphyloccus aureus
Lane 8: Enterococcus faecalis
Lane 9: Lactobacillus lactis
Lane 10: Bacillus subtilus
Lane 11: Listeria monocytogenes
Lane 12: Listeria innocua
Lane 13: Listeria murrayi
Lane 14: Listeria welshimeri
Lane 15: Listeria grayi
Lane 16: Mycobacterium bovis
Lane B: Molecular weight marker V
The PCR products generated using the genus-specific amplification described in this Example, and shown in
In
Lane A: Molecular weight marker V
Lane 1: E. coli
Lane 2: S. poona
Lane 3: K. aerogenes
Lane 4: P. mirabilis
Lane 5: P. rettgeri
Lane 6: A. hydrophilia
Lane 7: S. aureus
Lane 8: E. faecalis
Lane 9: L. lactis
Lane 10: B. subtilus
Lane 11: L. monocytogenes strain 1
Lane 12: L. monocytogenes strain 2
Lane 13: L. monocytogenes strain 3
Lane 14: L. monocytogenes strain 4
Lane 15: L. monocytogenes clinical isolate
Lane 16: L. innocua
Lane 17: L. murrayi
Lane 18: L. welshimeri
Lane 19: L. grayi
Lane 20: M. bovis
Lane B: Molecular weight marker V
Escherichia
coli
Salmonella
poona
Klebsiella
aerogenes
Proteus
mirabilis
Proteus
rettgeri
Aeromonas
hydrophilia
Staphyloccus
aureus
Entrococcus
aecalis
Lactobacillus
lactis
Bacillus
subtilus
Listeria
monocytogenes strain 1
Listeria
monocytogenes strain 2
Listeria
monocytogenes strain 3
Listeria
monocytogenes strain 4
Listeria
monocytogenes clinical isolate
Listeria
innocua
Listeria
murrayi
Listeria
welshimeri
Listeria
grayi
Mycobacterium
bovis
One of the typed L. monocytogenes strains, strain 4, failed to generate a positive signal with this probe. DNA sequencing of the PCR amplified ssrA gene from this strain demonstrated that it contained a probe target region identical to L. innocua. It should be noted however that the ssrA gene from this strain contains other regions where the sequence is identical to the previously characterised L. monocytogenes strain and that these sequences are different to the L. innocua sequence, as shown in
LGTm (A), LStm (B) and a Campylobacter upsaliensis 16S-23S rRNA spacer (C-5′ CATTAAACTTTAGCAAGGAAGTG 3′) SEQ ID NO: 228 oligonucleotide probe were irreversibly bound to nylon membrane strips and hybridised to with amplified ssrA PCR product, using the genus specific primers Ltm1 and Ltm2 (Ltm1 was labelled with biotin at the 5′ end), from L. monocytogenes (1-6), L. innocua (7-10), Z. ivanovii (11), L. murrayi (12), L. seeligeri (13), L. welshmeri (14) and L. grayii (15). The ssrA amplified PCR products, using tmU5′ and tmU3′ (tmU5′ was labelled with biotin at the 5′ end), were also hybridised to the nylon membrane strips from the Gram-positive bacteria, B. subtilus, L. lactis, S. aureus, S. epidermis, E. faecalis, C. perfringins (16-21) and the Gram-negative bacteria E. coli, S. enteritidis, P. Rettgeri, K. aerogenes (22-25). As shown in
Clustal W alignments as shown in
E. coli and L. monocytogenes cultures were heat treated at 80° C., for 20 min. in the case of E. coli and 40 min. in the case of L. monocytogenes and at 120° C. for 15 min. (autoclaving) after overnight growth and tested for viability at 0 h, 1 h, 2 h, 6 h, 12 h, 24 h and 48 h after heat treatment. No viability was observed at each time period tested. Total RNA was also isolated at these time periods and electrophoresed on denaturing 1.2% agarose gels and Northern blotted. Each blot was hybridised to, in the case of E. coli (
A 100 ml culture of L. monocytogenes was grown overnight in liquid culture. After growth, serial dilutions of the cells were carried out and viability was determined by spread plating on nutrient agar plates. Simultaneously, total RNA was isolated from a 1 ml aliquot of these cells. The remainder of the cells were heated at 65° C. for 20 min. Cells were then removed for both viability analysis and total RNA isolation. Samples were taken for viability and RNA isolation at time periods of 0 h, 2 h, 6 h and 24 h after treatment.
Spread plating on nutrient agar plates indicated that heat treatment killed L. monocytogenes cells, with no viable colony forming units observed. Each RNA sample isolated was then treated with DNase to remove any contaminating DNA and total RNA samples (100 ng) were subjected to Reverse Transcriptase-PCR amplification using the Listeria genus specific ssrA/tmRNA oligonucleotide primers Ltm1 and Ltm2. Negative control amplification reactions included primers, target, and Tag polymerase, but no Reverse Transcriptase. The results of the amplification reactions are shown in
Amplified tmRNA RT-PCR products were only observed with the RNA sample which was not heat treated. All other samples gave no RT-PCR product indicating that the tmRNA molecules in these samples may have been degraded in the non-viable heat treated cells.
In
| Number | Date | Country | Kind |
|---|---|---|---|
| PCT/IE99/00043 | May 1999 | WO | international |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/IE00/00066 | 5/15/2000 | WO | 00 | 1/31/2002 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO00/70086 | 11/23/2000 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 6348315 | Pluckthun et al. | Feb 2002 | B1 |
| 7115366 | Felden | Oct 2006 | B1 |
| 20060216733 | Felden | Sep 2006 | A1 |
| Number | Date | Country |
|---|---|---|
| A 0 395 292 | Oct 1990 | EP |
| A 9848008 | Oct 1998 | WO |
| WO 0059918 | Oct 2000 | WO |
