EP1809746A1

EP1809746A1 - Polynucleotides for the detection of listeria species

Info

Publication number: EP1809746A1
Application number: EP05761955A
Authority: EP
Inventors: Nancy Dallaire; Nancy Bourassa
Original assignee: Warnex Research Inc
Current assignee: Warnex Research Inc
Priority date: 2004-07-06
Filing date: 2005-07-06
Publication date: 2007-07-25
Also published as: CA2613159A1; AU2005259732A1; WO2006002546A1

Abstract

A system for the detection of a variety of Listeria species in a test sample is provided. The system comprises polynucleotide primers and probes for the amplification and detection of a portion of a Listeria ssrA gene. The primers and probes can be used in real time diagnostic assays for rapid detection of Listeria in a variety of situations. Kits comprising the primers and probes are also provided.

Description

POLYNUCLEOTIDES FOR THE DETECTION OF LISTERIA

SPECIES

FIELD OF THE INVENTION

The present invention relates to the field of detection of microbial contaminants. More specifically, the invention relates to the detection of contamination by Listeria spp.

BACKGROUND OF THE INVENTION

Listeria strains are responsible for a growing number of reported cases of food poisoning throughout the world. There are currently six known Listeria species: L. monocytogenes, L. innocua, L. ivanovii, L. seeligeri, L. grayi and L. welshimeri. L. murrayi, which was previously believed to be an additional species in this genus, has been shown to be a subspecies of L. grayi [Rocourt, J., et ah, Int. J. Systematic Bacteriology, 42:171-174 (1992)]. Within the Listeria genus only I,, monocytogenes and L. ivanovii are considered pathogens. Bacteria from this genus are found in a . variety of environments including, soil aqueous environments and animal carriers. As such, Listeria can enter food production systems easily and has been associated with foods such as raw milk, cheeses, ice cream, raw vegetables, raw meats, raw and cooked poultry and processed meat products. Infections by Listeria cause the sudden onset of fever, nausea, headache, gastrointestinal symptoms, and vomiting; which may be followed by meningitis, meningo-encephalitis, or septicaemia. In the case of pregnant women, symptoms of infection can include intra-uterine infections of the fetus that result in spontaneous abortion, still-birth, or a generally disseminated infection of the neonate [see, Rocourt, J and Cossart P (1997) 'Listeria monocytogenes', in Food microbiology: fundamentals and frontiers, MP Doyle, LR Beuchat, and TJ Montville, eds. American Society of Microbiology Press. Washington D. C. pp. 337-352]. In order to prevent Listeria infections, methods of detection can be utilized that identify the presence of the bacteria in food, prior to consumer availability and consumption. However, due to relatively quick rates of food spoilage, many detection techniques, which require long time periods, are not time and cost effective. For example, a number of detection technologies require the culturing of bacterial samples for time periods of up to eight days. In that time, however, the product being tested must be placed in circulation for purchase and consumption. Therefore, a system that can rapidly identify the presence of Listeria in food samples is desirable.

A variety of methods have been described for the detection of bacterial contaminants. One of these methods is the amplification of a specific nucleotide sequence using specific primers in a PCR assay. Upon completion of the amplification of a target sequence, the presence of an amplicon is detected using agarose gel electrophoresis. This method of detection, while being more rapid than traditional methods requiring culturing bacterial samples, is still relatively time consuming and subject to post-PCR contamination during the running of the agarose gel.

An additional technology utilized for detection of bacterial contamination, is nucleic acid hybridization. In such detection methodologies, the target sequence of interest is amplified and then hybridized to an oligonucleotide probe which possesses a complementary nucleic acid sequence to that of the target molecule. The probe can be modified so that detection of the hybridization product may occur, for example, the probe can be labelled with a radioisotope or fluorescent moiety.

U.S. Patent No. 5,922,538 describes a method of determining whether an unknown bacterium is a member either of the Listeria monocytogenes species or of the Listeria genus using specific DNA marker sequences. These marker sequences were identified from DNA fragments that were, in turn identified using a RAPD amplification protocol. The marker sequences are used for the design of primers and probes for the identification of members of the Listeria monocytogenes species or the Listeria genus.

A method of detecting and differentiating Listeria species is described by Bubert et al. [Appl. Environ. Microbiol.; 65:4688-4692 (1999)]. The method employs five different primers based on the iap gene sequence in a multiplex PCR reaction and can identify L. monocytogenes, L. grayi, L. innocua, L seeligeri, L. welshimeri and L. ivanovii species. Molecular beacons represent a powerful tool for the rapid detection of specific nucleotide sequences. Molecular beacons are capable of detecting the presence of a complementary nucleotide sequence even in homogenous solutions. Molecular beacons can be described as hairpin stem-and-loop oligonucleotide sequences, in which the loop portion of the molecule represents a probe sequence, which is complementary to a predetermined sequence in a target nucleic acid. On one arm of the beacon sequence is attached a fluorescent moiety while on the other arm of the beacon is a non-fluorescent quencher. The stem portion of the stem-and-loop sequence holds the two arms of the beacon in close proximity. Under these circumstances, the fluorescent arm of the oligonucleotide does not fluoresce. When the fluorescent and quencher arms are in close proximity, the quencher moiety receives energy from the fluorophore and dissipates such energy as heat, instead of light emission. Thus, the fluorophore is unable to fluoresce when the oligonucleotide is in the hairpin loop configuration. However, when the beacon encounters a nucleic acid sequence complementary to its probe sequence, the probe hybridizes to the nucleic acid sequence, forming a stable complex, hi the result, the arms of the probe are separated and the fluorophore is allowed to emit light. The emission of light is indicative of the presence of the specific nucleic acid sequence. Individual molecular beacons are highly specific for the DNA sequences they are complementary to.

SsrA RNA, also known as tmRNA and 10Sa RNA, is the product of the ssrA gene, which has been found in a wide variety of bacterial species. SsrA RNA is involved in the degradation of incomplete polypeptides. SsrA RNA mediates the addition of a peptide tag to the C-terminus of an incomplete polypeptide when the ribosome stalls as a result the mRNA lacking a properly functioning stop codon in a process known as trans-translation. The tagged polypeptide is then directed for proteolysis, hi addition the SsrA RNA reduces the synthesis of incomplete polypeptides by facilitating the degradation of the defective mRNA molecules. [Atkins JF₅ Gesteland RF. Nature 1996, 379:769-771; Jentsch S. Science 1996, 271:955-956; Keiler KC, et al Science 1996, 271:990-993; Komine Y, et al. Pt -oc Natl Acad Sd USA 199 A, 91:9223-9227; Williams KP, et al. EMBO J. 1999, 18:5423-5433; Williams KP. Nucleic Acids Res. 2000, 28:168; Yamamoto Y, et al. RNA 2003, 9:408-418]. The use of sequences of the ssrA gene for the detection of Listeria and other organisms has been previously described in International Patent Applications PCTYFROl/03061 (WO02/28891) and PCT/IEOO/00066 (WO00/70086). The latter patent application describes two primers and a probe that are "genus specific" for Listeria. These "genus specific" primers were used in a standard PCR reaction and shown to be capable of amplifying a 260 bp fragment of the ssrA gene from four species of Listeria (L. monocytogenes, L. innocua, L. grayi (L. murrayi) and L. welshimeri). Similarly, the "genus specific" probe was shown to be capable of detecting a fragment of the ssrA gene of approximately 350 bp from these four Listeria species in a standard Southern blot procedure. The primers and probe correspond to regions that are completely conserved across these four species of Listeria and that show the greatest difference to the ssrA gene sequence of Bacillus subtilis.

Schδnhuber W, et al. [BMC Microbiol. 1:20 (2001)] describe the use of tmRNA for bacterial identification and particularly, the use of tmRNA to distinguish between species within a genus. Schδnhuber demonstrates that the most highly conserved regions of the ssrA gene product are at the 3' and 5' ends and that the tmRNA nucleotide sequences of some bacteria, such as Listeria, display considerable divergence between species, in particular within the internal region of the ssrA gene.

This background information is provided for the purpose of making known information believed by the applicant to be of possible relevance to the present invention. No admission is necessarily intended, nor should be construed, that any of the preceding information constitutes prior art against the present invention.

SUMMARY OF THE INVENTION

An object of the present invention is to provide polynucleotides for the detection of Listeria species. In accordance with one aspect of the present invention, there is provided a combination of polynucleotides for amplification and detection of one or more target nucleotide sequences from a Listeria ssrA gene, said combination selected from the group of: (a) a combination comprising a first polynucleotide primer comprising at least 7 consecutive nucleotides of the sequence as set forth in any one of SEQ DD NOs:2-15; a second polynucleotide primer comprising at least 7 consecutive nucleotides of a sequence complementary to any one of SEQ ID NOs:2-15 and a first polynucleotide probe comprising at least 7 consecutive nucleotides of the sequence as set forth in SEQ ID NO: 16, or the complement thereof;

(b) a combination comprising a first polynucleotide primer comprising at least 7 consecutive nucleotides of the sequence as set forth in any one of SEQ ID NOs:28-41; a second polynucleotide primer comprising at least 7 consecutive nucleotides of a sequence complementary to any one of SEQ ID NOs:28-41, and a polynucleotide probe comprising at least 7 consecutive nucleotides of the sequence as set forth in SEQ ID NO:42, or the complement thereof;

(c) a combination comprising a first polynucleotide primer comprising at least 7 consecutive nucleotides of the sequence as >set forth in any one of SEQ ID NOs:50-63; a second polynucleotide primer comprising at least 7 consecutive nucleotides of a sequence complementary to any one of SEQ ID NOs:50-63, and a polynucleotide probe comprising at least 7 consecutive nucleotides of the sequence as set forth in SEQ ID NO: 64, or the complement thereof, and

(d) a combination comprising two or more of the combinations of (a), (b)

I and (c).

In accordance with another aspect of the present invention, there is provided a method of detecting Listeria in a sample, said method comprising the steps of: (i) contacting a sample suspected of containing, or known to contain, one or more Listeria target nucleotide sequences with a combination of polynucleotides of the invention under conditions that permit amplification and detection of said target nucleotide sequence(s), and (U) detecting any amplified target sequence(s), wherein detection of an amplified target sequence indicates the presence of Listeria in the sample. In accordance with another aspect of the present invention, there is provided a kit for the detection of Listeria in a sample, said kit comprising a combination of polynucleotides selected from the group of:

(a) a combination comprising a first polynucleotide primer comprising at least 7 consecutive nucleotides of the sequence as set forth in any one of SEQ E) NOs:2-15; a second polynucleotide primer comprising at least 7 consecutive nucleotides of a sequence complementary to any one of SEQ ID NOs:2-15 and a first polynucleotide probe comprising at least 7 consecutive nucleotides of the sequence as set forth in SEQ ID NO: 16, or the complement thereof;

(c) a combination comprising a first polynucleotide primer comprising at least 7 consecutive nucleotides of the sequence as set forth in any one of SEQ ID NOs:50-63; a second polynucleotide primer comprising at least 7 consecutive nucleotides of a sequence complementary to any one of SEQ ID NOs:50-63, and a polynucleotide probe comprising at least 7 consecutive nucleotides of the sequence as set forth in SEQ ID NO: 64, or the complement thereof, and

(d) a combination comprising two or more of the combinations of (a), (b) and (c).

In accordance with another aspect of the present invention, there is provided a pair of polynucleotide primers for amplification of a portion of a Listeria ssrA gene, said portion being less than 250 nucleotides in length and comprising at least 50 consecutive nucleotides of the sequence set forth in any one of SEQ ID NOs: 16, 42 or 64, said pair of polynucleotide primers comprising: (a) a first polynucleotide primer comprising at least 7 consecutive nucleotides of the sequence as set forth in SEQ ID NO:1; and (b) a second polynucleotide primer comprising at least 7 consecutive nucleotides of a sequence complementary to SEQ ID NO:1.

In accordance with another aspect of the present invention, there is provided an isolated Listeria specific polynucleotide consisting essentially of: (a) the sequence as set forth in SEQ ID NO:16, SEQ ID NO:42 or SEQ ID NO:64, or a fragment of said sequence, or (b) a sequence that is the complement of (a).

In accordance with another aspect of the present invention, there is provided a polynucleotide primer of between 7 and 100 nucleotides in length for the amplification of a portion of a Listeria ssrA gene, said polynucleotide primer comprising at least 7 consecutive nucleotides of the sequence as set forth in SEQ ID NO: 16, SEQ ID NO:42 or SEQ ID NO:64, or the complement thereof.

Li accordance with another aspect of the present invention, there is provided a polynucleotide probe of between 7 and 100 nucleotides in length for detection of Listeria, said polynucleotide probe comprising at least 7 consecutive nucleotides of the sequence as set forth in SEQ ID NO: 16, SEQ ID NO:42 or SEQ ID NO:64, or the complement thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features of the invention will become more apparent in the following detailed description in which reference is made to the appended drawings wherein:

Figure 1 presents a multiple alignment using the coding strand of the ssrA gene from a number of Listeria strains [SEQ ID NOs:2-15] and shows conserved regions of the gene. Shaded blocks highlight the following regions: bases 31 to 53 - forward primer #1 [SEQ E) NO: 18]; bases 72 to 90 - binding site for molecular beacon probe #1 [SEQ ID NO: 20]; bases 100 to 117 - binding site for reverse primer #1 [SEQ ID NO: 19]; Figure 2 presents the arrangement in one embodiment of the invention of PCR primers and molecular beacon on a portion of the ssrA gene sequence. Numbers in parentheses indicate the positions of the first and last nucleotides of each feature of the PCR product generated with primers SEQ ID NOs: 18 and 19;

Figure 3 presents the secondary structure of a molecular beacon [SEQ ID NO: 20] in accordance with one embodiment of the invention;

Figure 4 presents a multiple alignment showing conserved regions of a portion of the coding strand of the ssrA gene from various Listeria strains [SEQ ID NOs:28-41]. Shaded blocks highlight the following regions: bases 145 to 165 - forward primer #3 [SEQ ID NO:44]; bases 205 to 228 - binding site for molecular beacon #3 [SEQ ID NO: 46]; bases 240 to 264 - binding site for reverse primer #3 [SEQ ID NO: 45];

Figure 5 presents the arrangement in one embodiment of the invention of PCR primers and molecular beacon on a portion of the ssrA gene sequence. Numbers in parentheses indicate the positions of the first and last nucleotides of each feature of the PCR product generated with primers SEQ ID NOs:44 and 45;

Figure 6 presents the secondary structure of a molecular beacon [SEQ ID NO: 46] in accordance with one embodiment of the invention;

Figure 7 presents a multiple alignment using the non-coding strand of the ssrA gene from a number of Listeria strains [SEQ ID NOs:50-63] and shows conserved regions of the gene. Shaded blocks highlight the following regions: bases 66 to 87 forward primer #5[SEQ ID NO: 72]; bases 133 to 154 - binding site for molecular beacon #4 [SEQ ID NO: 68]; bases 164 to 181 - binding site for reverse primer [SEQ ID NO: 74];

Figure 8 presents the arrangement in one embodiment of the invention of PCR primers and molecular beacon on a portion of the ssrA gene sequence. Numbers in parentheses indicate the positions of the first and last nucleotides of each feature of the PCR product generated with primers SEQ ID NOs:72 and 74; Figure 9 presents the secondary structure of a molecular beacon [SEQ ID NO: 68] in accordance with one embodiment of the invention;

Figure 10 presents (A) the sequence of a Listeria ssrA target sequence [SEQ ID NO:1] (coding strand) comprising three ssrA consensus sequences identified in one embodiment of the invention, (B) the sequence of a ssrA consensus sequence (coding strand) [SEQ ID NO:16], (C) the sequence of a highly conserved region [SEQ ID NO: 17] identified within the consensus sequence shown in (B), (D) the sequence of a second ssrA consensus sequence [SEQ ID NO:42] (coding strand), (E) the sequence of a highly conserved region [SEQ ID NO:43] identified within the consensus sequence shown in (D), (F) the sequence of a third consensus region^~[SEQ ID NO:64] (non coding strand), and (G) the sequence of a highly conserved region [SEQ ID NO:65] identified within the consensus sequence shown in (F). The following abbreviations are used to represent multiple bases: Y = T or C; R = A or G; N = A, G, C or T; M = A or C; W = A or T; K = G or T; and

Figure 11 presents a multiple alignment using the coding strand of the ssrA gene from a number of Listeria strains [SEQ ID NOs:28-41] showing the three overlapping consensus sequences [SEQ ID NOs: 16, 42 and 64].

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides for a system for the detection of Listeria species in a test sample. The system comprises polynucleotides designed to amplify and/or detect one or more Listeria target nucleotide sequences. In the context of the present invention, a target nucleotide sequence is a nucleic acid sequence that corresponds to an internal portion of the Listeria ssrA gene and that comprises one or more conserved regions (consensus sequences). In a specific embodiment of the present invention, the target nucleotide sequence is less than 250 nucleotides in length and comprises between one and three overlapping consensus sequences. Unexpectedly, although being present in the internal region of the ssrA gene and thus encompassing less well conserved regions of the gene [see, Schδnhuber W, et al, BMC Microbiol. 1:20 (2001)], the target sequence identified by the present invention can be used to design primers and probes that are demonstrated to be capable of amplifying and detecting various species from the Listeria genus. The system of the present invention is well suited for use in real-time amplification/detection methodologies, thereby providing for rapid detection of various Listeria species in test samples.

The present invention thus provides for primer and probe polynucleotides that are capable of amplifying and/or detecting a Listeria target sequence and, as such, are capable of detecting a variety of Listeria species. It will be understood by those skilled in the art that detection of all known species of Listeria (of which there are currently six) may not be necessary in all situations. For example, the species Listeria grayi is found only infrequently as a contaminant of foodstuffs. Primers/probes capable of detecting L. monocytogenes, L. innocua, L. ivanovii, L. seeligeri and L. welshimeri, therefore, are suitable for detecting Listeria contamination of foods and environmental samples. Thus, one embodiment of the invention provides for polynucleotide primers/probes that are capable of detecting strains from at least five different species of Listeria. These primers/probes are designated "primary" primers/probes. Primary primers/probes can be capable of detecting five out of the six currently known species of Listeria, or they can be capable of detecting all six of the currently known species of Listeria.

The system provided by the present invention can be designed to detect at least five species of Listeria or to detect all six presently known species of Listeria depending on the particular primer/probe sequences selected. For example, the system can comprise primary primers and probes capable of detecting five species of Listeria together with a second set of primary primers/probes that are capable of detecting a different combination of Listeria species such that the combination provides for detection of all six known species of Listeria. Alternatively, the system can comprise a combination of two sets of primary primers/probes, a first set capable of detecting all species of Listeria with a low detectable signal, and a second set capable of detecting five or more species of Listeria with a high detectable signal. The present invention also contemplates that the system can further comprise a set of secondary primers/probes, wherein the secondary primers/probes are capable of detecting the species of Listeria not detected by the primary primers/probes. In accordance with one embodiment of the present invention, the primary primers and probes of the invention demonstrate a sensitivity for the detection of strains of Listeria of at least 90%, as defined herein, wherein the strains represent at least five species of Listeria. In another embodiment, the primers and probes demonstrate a sensitivity of at least 92%. In further embodiments, the primers and probes demonstrate a sensitivity of at least 94%.

In addition, both the primary and secondary primers and probes of the invention demonstrate a specificity for Listeria target nucleotide sequences of at least 95%, as defined herein. In one embodiment, the primers and probes of the invention demonstrate a specificity for Listeria target nucleotide sequences of at least 97%. In another embodiment, the primers and probes of the invention demonstrate a specificity for Listeria target nucleotide sequences of at least 98%. In further embodiments, the primers and probes of the invention demonstrate a specificity for Listeria target nucleotide sequences of at least 99%, and at least 99.5%.

The present invention provides for systems comprising combinations of primers and/or probes that target the same or different ssrA target sequences. As indicated above, systems comprising combinations of more than one set of primers/probes can increase the sensitivity of detection, increase the amounts of detectable signal produced, increase the specificity, and the like, over either set alone.

Thus, in one embodiment, the present invention provides for a system comprising a combination of sets of primers/probes that target different ssrA consensus sequences. In another embodiment, the present invention provides for a system comprising a combination of primers/probes that target the same ssrA consensus sequence. In a further embodiment, the present invention provides for a system comprising a combination of sets of polynucleotides, wherein each set comprises a primer pair and a probe, and wherein the combination demonstrates a greater sensitivity in detecting Listeria species than each set of primers and probe alone. In another embodiment, a system is provided that comprises a combination of primers and probes that demonstrates a sensitivity of at least 95%. In another embodiment, there is provided a system that comprises a combination of primers and probes that demonstrates a sensitivity of at least 96%. In further embodiments, there are provided systems that comprise combinations of primers and probes that demonstrate a sensitivity of at least 97%, at least 98% and at least 99%.

The polynucleotide primers and probes of the present invention are suitable for use in detecting the presence of various Listeria species in a sample, such as a clinical sample, microbiological pure culture, or a sample related to food, environmental or pharmaceutical quality control processes. The present invention contemplates methods and assays for detecting Listeria species in a sample using one or more polynucleotides targeting a single consensus sequence, as well as methods and assays using combinations of polynucleotides that target different consensus sequences. In one embodiment, the invention provides diagnostic assays that can be carried out in real time and addresses the need for rapid detection of Listeria species in a variety of biological samples.

Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The terms "oligonucleotide" and "polynucleotide" as used interchangeably in the present application refer to a polymer of greater than one nucleotide in length of ribonucleic acid (RNA), deoxyribonucleic acid (DNA), hybrid RNA/DNA, modified RNA or DNA, or RNA or DNA mimetics. The polynucleotides may be single- or double-stranded. The terms include polynucleotides composed of naturally-occurring nucleobases, sugars and covalent internucleoside (backbone) linkages as well as polynucleotides having non-naturally-occurring portions which function similarly. Such modified or substituted polynucleotides are well-known in the art and for the purposes of the present invention, are referred to as "analogues."

The terms "primer," "polynucleotide primer" and "amplification primer," as used herein, refer to a short, single-stranded polynucleotide capable of hybridizing to a complementary sequence in a nucleic acid sample. A primer serves as an initiation point for template-dependent nucleic acid synthesis. Nucleotides are added to a primer by a nucleic acid polymerase, which adds such nucleotides in accordance with the sequence of the template nucleic acid strand. A "primer pair" or "primer set" refers to a set of primers including a 5 ' upstream primer that hybridizes with the 5 ' end of the sequence to be amplified and a 3' downstream primer that hybridizes with the complementary 3 ' end of the sequence to be amplified. The term "forward primer" as used herein, refers to a primer which anneals to the 5' end of the sequence to be amplified. The term "reverse primer", as used herein, refers to a primer which anneals to the complementary 3 ' end of the sequence to be amplified.

The terms "probe" and "polynucleotide probe," as used herein, refer to a polynucleotide used for detecting the presence of a specific nucleotide sequence in a sample. Probes specifically hybridize to a target nucleotide sequence, or the complementary sequence thereof, and may be single- or double-stranded.

The term "specifically hybridize," as used herein, refers to the ability of a polynucleotide to bind detectably and specifically to a target nucleotide sequence. Polynucleotides, oligonucleotides and fragments thereof specifically hybridize to target nucleotide sequences under hybridization and wash conditions that minimize appreciable amounts of detectable binding to non-specific nucleic acids. High stringency conditions can be used to achieve specific hybridization conditions as is known in the art. Typically, hybridization and washing are performed at high stringency according to conventional hybridization procedures and employing one or more washing step in a solution comprising 1-3 x SSC, 0.1-1% SDS at 50-70⁰C for 5- 30 minutes.

The term "specificity," as used herein, refers to the ability of a primer or primer pair to amplify, or a probe to detect, nucleic acid sequences from Listeria but not from other bacterial species. "% specificity" is defined by a negative validation test wherein the primers and/or probe are tested against a panel of at least 100 bacterial species other than Listeria. Thus, for example, a pair of primers that does not amplify any nucleic acid sequences from the panel of bacterial species would be defined as demonstrating 100% specificity, a pair of primers that amplified a nucleic acid sequence from one bacterial species in a panel of 100 species would be defined as demonstrating 99% specificity, a pair of primers that amplified a nucleic acid sequence from two different bacterial species in a panel of 100 species would be defined as demonstrating 98% specificity, etc.

The term "sensitivity," as used herein, refers to the ability of a primer or primer pair to amplify, or a probe to detect, nucleic acid sequences from a range of Listeria strains. "% sensitivity" is defined by a positive validation test wherein the primers and/or probe are tested against a panel of at least 50 Listeria strains from at least five different Listeria species. Thus, for example, a pair of primers that amplifies nucleic acid sequences from all Listeria strains in the panel would be defined as demonstrating 100% sensitivity, a pair of primers that amplified nucleic acid sequences from 49 Listeria strains in a panel of 50 strains would be defined as demonstrating 99% sensitivity, a pair of primers that amplified nucleic acid sequences from 48 Listeria strains in a panel of 50 strains would be defined as demonstrating 98% sensitivity, etc.

The term "corresponding to" refers to a polynucleotide sequence that is identical to all or a portion of a reference polynucleotide sequence, hi contradistinction, the term "complementary to" is used herein to indicate that the polynucleotide sequence is identical to all or a portion of the complementary strand of a reference polynucleotide sequence. For illustration, the nucleotide sequence "TATAC" corresponds to a reference sequence "TATAC" and is complementary to a reference sequence "GTATA."

The terms "hairpin" or "hairpin loop" refer to a single strand of DNA or RNA, the ends of which comprise complementary sequences, whereby the ends anneal together to form a "stem" and the region between the ends is not annealed and forms a "loop." Some probes, such as molecular beacons, have such "hairpin" structure when not hybridized to a target sequence. The loop is a single-stranded structure containing sequences complementary to the target sequence, whereas the stem self-hybridises to form a double-stranded region and is typically unrelated to the target sequence. Nucleotides that are both complementary to the target sequence and that can self- hybridise can be included in the stem region.

The terms "target sequence" or "target nucleotide sequence," as used herein, refer to a particular nucleic acid sequence in a test sample to which a primer and/or probe is intended to specifically hybridize. A "target sequence" is typically longer than the primer or probe sequence and thus can contain multiple "primer target sequences" and "probe target sequences." A target sequence may be single or double stranded. The term "primer target sequence" as used herein refers to a nucleic acid sequence in a test sample to which a primer is intended to specifically hybridize. The term "probe target sequence" refers to a nucleic acid sequence in a test sample to which a probe is intended to specifically hybridize.

As used herein, the term "about" refers to a +/-10% variation from the nominal value. It is to be understood that such a variation is always included in any given value provided herein, whether or not it is specifically referred to.

LISTERIA TARGETSEQUENCE

As indicated above, the Listeria target nucleotide sequence provided by the present invention corresponds to an internal portion of the Listeria ssrA gene and comprises one or more consensus sequences. In order to identify a suitable region of the Listeria ssrA gene that could serve as a target sequence for the design of specific primers and probes for the detection of Listeria species, Listeria ssrA gene sequences were subjected to a multiple alignment analysis. Exemplary multiple sequence alignments of portions of the ssrA gene from various Listeria species and strains are shown in Figures 1, 4, 7 and 11.

A target nucleotide sequence (SEQ ID NO:1, shown in Figure 10A) comprising three overlapping consensus sequences was thus identified in the ssrA gene. The consensus sequences are as follows: an 87 nucleotide region (SEQ ID NO: 16; shown in Figure 10B), a 119 nucleotide region (SEQ ID NO:42; shown in Figure 10D) and a 115 nucleotide region (SEQ ID NO:64; shown in Figure 10F). These sequences are referred to herein as ssrA consensus sequences. Accordingly in one embodiment, the present invention provides a target nucleotide sequence of less than 250 nucleotides in length that comprises three overlapping consensus sequences, wherein the consensus sequences consist of SEQ ID NO: 16, SEQ ID NO: 42 and SEQ ID NO: 64. hi a specific embodiment, the present invention provides a target nucleotide sequence consisting of SEQ ID NO:1 (shown in Figure 10A), or the complement thereof, hi another embodiment, the present invention provides an isolated polynucleotide specific for Listeria species consisting of one or more of the consensus sequences set forth in any one of SEQ ID NOs: 16, 42 or 64, or the complement thereof, that can be used as a target sequence for the design of primers and/or probes for the specific detection of Listeria species.

hi accordance with the present invention, the target nucleic acid sequence can comprise all, or a portion, of one or more of the consensus sequences set forth in any one of SEQ ID NOs:16, 42 or 64. Thus, one embodiment of the invention provides for a target sequence suitable for the specific detection of Listeria comprising at least 60% of the sequence set forth in any one of SEQ ID NOs: 16, 42 or 64, or the complement thereof. In another embodiment, the target sequence comprises at least 75% of the sequence set forth in any one of SEQ ID NOs:16, 42 or 64, or the complement thereof, hi a further embodiment, the target sequence comprises at least 80% of the sequence set forth in any one of SEQ ID NOs: 16, 42 or 64, or the complement thereof. Target sequences comprising at least 85%, 90%, 95% and 98% of the sequence set forth in any one of SEQ ID NOs: 16, 42 or 64, or the complement thereof, are also contemplated.

Alternatively, such portions of the consensus sequence can be expressed in terms of consecutive nucleotides of the sequence set forth in any one of SEQ ID NOs:16, 42 or 64. Accordingly, target sequences comprising portions of the consensus sequence including at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, and at least 85 consecutive nucleotides of the sequence set forth in any one of SEQ ID NOs: 16, 42 or 64, or the complement thereof, are contemplated. By "at least 50 consecutive nucleotides" it is meant that the target sequence may comprise any number of consecutive nucleotides between 50 and the full length of the sequence set forth in any one of SEQ ID NOs: 16, 42 or 64 {i.e. 87, 119 or 115 nucleotides, respectively), thus this range includes portions of the consensus sequence that comprise at least 51, at least 52, at least 53, at least 54, etc, consecutive nucleotides of the sequence set forth in any one of SEQ ID NOs: 16, 42 or 64, or the complement thereof.

As indicated above, the present invention also contemplates target sequences comprising two or more consensus sequences, or portions thereof as described above, for the specific detection of Listeria species. Thus, for example, the target sequence can span a region of the ssrA gene sequence between the region corresponding/ complementary to SEQ ID NO: 16 and the region corresponding/complementary to SEQ ID NO:64 and include at least 50 consecutive nucleotides of SEQ ID NO:16 and at least 50 consecutive nucleotides of SEQ ID NO:64. Alternatively, a target sequence can span a region of the ssrA gene sequence between the region corresponding/ complementary to SEQ ID NO: 64 and the region corresponding/complementary to SEQ ID NO:42 and include at least 50 consecutive nucleotides of SEQ ID NO:64 and at least 50 consecutive nucleotides of SEQ ID NO:42. A target sequence could also span a region of the ssrA gene sequence between the region corresponding/ complementary to SEQ ID NO: 16 and the region corresponding/complementary to SEQ ID NO:42 including at least 50 consecutive nucleotides of SEQ ID NO: 16, at least 50 consecutive nucleotides of SEQ ID NO:42 and encompassing all of SEQ ID NO:64. In a specific embodiment of the present invention, the target sequence comprises at least 50 nucleotides of SEQ ID NO: 64.

Within the identified consensus sequences, additional highly conserved regions were identified. The highly conserved region identified within the ssrA consensus sequence shown in SEQ ID NO: 16 is 19 nucleotides in length and has a sequence corresponding to SEQ ID NO:17 (as shown in Figure 10C). The highly conserved region identified within the ssrA consensus sequence shown in SEQ ID NO:42 is 24 nucleotides in length and has a sequence corresponding to SEQ ID NO:43 (as shown in Figure 1 OE) and the highly conserved region identified within the ssrA consensus sequence shown in SEQ DD NO: 64 is 22 nucleotides in length and has a sequence corresponding to SEQ ID NO:65 (as shown in Figure 10G). Accordingly, one embodiment of the present invention provides for target sequences that comprise all or a portion of a sequence corresponding to any one of SEQ ID NO: 17, 43 or 65, or the complement thereof. Target sequences comprising both SEQ ID NOs: 17 and 43, SEQ ID NOs:17 and 65, SEQ ID NOs:65 and 43, or portions thereof, are also contemplated.

It will also be appreciated that the target sequence may include additional nucleotide sequences that are found upstream and/or downstream of the consensus sequence(s) in the Listeria genome. As the assays provided by the present invention typically include an amplification step, it may be desirable to select an overall length for the target sequence such that the assay can be conducted fairly rapidly. Thus, the target sequence typically has an overall length of less than about 500 nucleotides. In one embodiment, the target sequence has an overall length of less than about 400 nucleotides. Pn another embodiment, the target sequence has an overall length of less than about 300 nucleotides. In a further embodiment, the target sequence has an overall length of less than 260 nucleotides. In other embodiments, the target sequence has an overall length of less than about 250, less than about 225, less than about 200, less than about 175, less than about 150 and less than about 130 nucleotides. In a further embodiment, the target sequence has an overall length corresponding to the length of a consensus sequence, i.e. 87, 119 or 115 nucleotides.

POLYNUCLEOTIDE PRIMERS AND PROBES

The system of the present invention comprises polynucleotides for the amplification and/or detection of one or more Listeria target nucleotide sequences in a sample. The polynucleotide primers and probes of the invention comprise a sequence that corresponds to or is complementary to the portion of a Listeria ssrA gene and are capable of specifically hybridizing to Listeria nucleic acids. In one embodiment, the polynucleotides of the invention comprise a sequence that corresponds to or is complementary to a portion of a Listeria ssrA gene sequence as set forth in SEQ ID NO:1. In another embodiment, the polynucleotides of the invention comprise a sequence that corresponds to or is complementary to a portion of a Listeria ssrA gene sequence as set forth in any one of SEQ ID NOs:2-15, 28-41 and 50-63. In a further embodiment, the polynucleotides of the invention comprise a sequence that corresponds to or is complementary to a portion of a Listeria ssrA gene sequence as set forth in any one of SEQ ID NOs:5-15, 31-41 and 53-63.

The polynucleotide primers and probes of the present invention are generally between about 7 and about 100 nucleotides in length. One skilled in the art will understand that the optimal length for a selected polynucleotide will vary depending on its intended application {i.e. primer, probe or combined primer/probe) and on whether any additional features, such as tags, self-complementary "stems" and labels (as described below), are to be incorporated. In one embodiment of the present invention, the polynucleotides are between about 10 and about 100 nucleotides in length, hi another embodiment, the polynucleotides are between about 12 and about 100 nucleotides in length. In other embodiments, the polynucleotides are between about 12 and about 50 nucleotides and between 12 and 35 nucleotides in length.

One skilled in the art will also understand that the entire length of the polynucleotide primer or probe does not need to correspond to or be complementary to its target sequence within the Listeria ssrA gene in order to specifically hybridize thereto. Thus, the polynucleotide primers and probes can comprise nucleotides at the 5' and/or 3 ' termini that are not complementary to the primer target sequence. Such non- complementary nucleotides may provide additional functionality to the primer/probe, for example, they may provide a restriction enzyme recognition sequence or a "tag" that facilitates detection, isolation or purification. Alternatively, the additional nucleotides may provide a self-complementary sequence that allows the primer/probe to adopt a hairpin configuration. Such configurations are necessary for certain probes, for example, molecular beacon and Scorpion probes.

The present invention also contemplates that one or more position within the polynucleotide can be degenerate, i.e. can be filled by one of two or more alternate nucleotides. As is known in the art, certain positions in a gene can vary in the nucleotide that is present at that position depending on the strain of bacteria that the gene originated from. By way of example, position 217 of the alignment shown in Figure 4 can contain a thymine ("T") nucleotide or a cytosine ("C") nucleotide depending on which strain of Listeria the ssrA gene originates from. Thus, a "degenerate" primer or probe designed to target this sequence can contain either a T or a C at the position corresponding to position 217 in the alignment. Such a degenerate primer or probe is typically prepared by synthesising a "pool" of polynucleotide primers or probes that contains, for example, approximately equal amounts of a polynucleotide containing a T at the degenerate position and a polynucleotide containing a C at the degenerate position.

Typically, the polynucleotide primers and probes of the invention comprise a sequence of at least 7 consecutive nucleotides that correspond to or are complementary to a portion of the Listeria ssrA gene sequence. As is known in the art, the optimal length of the sequence corresponding or complementary to the Listeria ssrA gene sequence will be dependent on the specific application for the polynucleotide, for example, whether it is to be used as a primer or a probe and, if the latter, the type of probe. Optimal lengths can be readily determined by the skilled artisan.

In one embodiment, the polynucleotides comprise at least 10 consecutive nucleotides corresponding or complementary to a portion of the Listeria ssrA gene sequence. In another embodiment, the polynucleotides comprise at least 12 consecutive nucleotides corresponding or complementary to a portion of the Listeria ssrA gene sequence. In a further embodiment, the polynucleotides comprise at least 14 consecutive nucleotides corresponding or complementary to a portion of the Listeria ssrA gene sequence. Polynucleotides comprising at least 16 and at least 18 consecutive nucleotides corresponding or complementary to a portion of the Listeria ssrA gene sequence are also contemplated.

Sequences of exemplary polynucleotides of the invention are set forth in Table 1. Further non-limiting examples for the polynucleotides of the invention include polynucleotides that comprise at least 7 consecutive nucleotides of any one of SEQ ID NOs:18, 19, 21, 23, 24, 25, 44, 45, 47, 49, 66, 67, 69, 71, 72, 73 or 74. Table 1 : Exemplary polynucleotides of the invention

¹ Y represents T or C

²WrepresentsAorT

³MrepresentsAorC

Primers

As indicated above, the polynucleotide primers of the present invention comprise a sequence that corresponds to or is complementary to a portion of the Listeria ssrA gene sequence. In accordance with the invention, the primers are capable of amplifying a Listeria target nucleotide sequence, wherein the target sequence comprises all or a portion of one or more ssrA consensus sequences, as described above.

Accordingly, in one embodiment, the present invention provides for primer pairs capable of amplifying a Listeria target nucleotide sequence, wherein the target sequence is less than about 500 nucleotides in length and comprises at least 50 consecutive nucleotides of any one of SEQ ID NOs: 16, 42 or 64, or the complement thereof, hi another embodiment, the present invention provides for primer pairs capable of amplifying a Listeria target nucleotide sequence, wherein the target sequence is less than about 500 nucleotides in length and comprises at least 50 consecutive nucleotides of both SEQ ID NOs: 16 and 64, or the complementary sequences thereof. In a further embodiment, the present invention provides for primer pairs capable of amplifying a Listeria target nucleotide sequence, wherein the target sequence is less than about 500 nucleotides in length and comprises at least 50 consecutive nucleotides of both SEQ ID NOs:64 and 42, or the complementary sequences thereof. In another embodiment, the present invention provides for primer pairs capable of amplifying a Listeria target nucleotide sequence, wherein the target sequence is less than about 500 nucleotides in length and comprises at least 50 consecutive nucleotides of both SEQ ID NOs:16 and 42, or the complementary sequences thereof.

m a specific embodiment, the present invention provides for primer pairs capable of amplifying a. Listeria target nucleotide sequence of less than 250 nucleotides in length that comprises at least 50 consecutive nucleotides of SEQ ID NO:64, or the complementary sequence thereof.

Thus, pairs of primers can be selected that comprise a forward primer corresponding to a portion of the Listeria ssrA gene sequence upstream of or within the region of the gene corresponding to the consensus sequence SEQ ID NO: 16 and a reverse primer that it is complementary to a portion of the Listeria ssrA gene sequence downstream of or within the region of the gene corresponding to SEQ ID NO:16. Similarly, pairs of primers can be selected that comprise a forward primer corresponding to a portion of the Listeria ssrA gene upstream of or within the region corresponding to the consensus sequence SEQ ID NO: 64 and a reverse primer that it is complementary to a portion of the Listeria ssrA gene downstream of or within the region corresponding to SEQ ID NO:64. Pairs of primers can be selected that comprise a forward primer corresponding to a portion of the Listeria ssrA gene upstream of or within the region corresponding to the consensus sequence SEQ ID NO:42 and a reverse primer that it is complementary to a portion of the Listeria ssrA gene downstream of or within the region corresponding to SEQ ID NO:42.

Similarly, for target sequences comprising more than one consensus sequence, pairs of primers can be selected that comprise a forward primer corresponding to a portion of the Listeria ssrA gene sequence upstream of or within the region of the gene corresponding to the consensus sequence SEQ ID NO: 16 and a reverse primer that it is complementary to a portion of the Listeria ssrA gene sequence downstream of or within the region of the gene corresponding to the consensus sequence SEQ ID NO:64; a forward primer corresponding to a portion of the Listeria ssrA gene sequence upstream of or within the region of the gene corresponding to the consensus sequence SEQ ID NO:64 and a reverse primer that it is complementary to a portion of the Listeria ssrA gene sequence downstream of or within the region of the gene corresponding to the consensus sequence SEQ ID NO:42, or a forward primer corresponding to a portion of the Listeria ssrA gene sequence upstream of or within the region of the gene corresponding to the consensus sequence SEQ ID NO: 16 and a reverse primer that it is complementary to a portion of the Listeria ssrA gene sequence downstream of or within the region of the gene corresponding to the consensus sequence SEQ ID NO:42.

In accordance with the present invention, the primers comprise at least 7 consecutive nucleotides of the ssrA gene sequence as set forth in SEQ ID NO:1, or the complement thereof. In one embodiment, the primers comprise at least 7 consecutive nucleotides of the sequence as set forth in any one of SEQ ID NOs:2-15, 28-41 and 50-63, or the complement thereof. In a further embodiment, the primers comprise at least 7 consecutive nucleotides of the sequence as set forth in any one of SEQ ID NOs:5-15, 31-41 and 53-63, or the complement thereof. In another embodiment, the primers comprise at least 7 consecutive nucleotides of the sequence set forth in any one of SEQ ID NOs: 16, 42 or 64, or the complement thereof.

Appropriate primer pairs can be readily determined by a worker skilled in the art. In general, primers are selected that specifically hybridize to a portion of the Listeria ssrA gene sequence without exhibiting significant hybridization to non-ssrA nucleic acids. In addition, the primers are selected to contain minimal sequence repeats and such that they show the least likelihood of dimer formation, cross dimer formation, hairpin structure formation and cross priming. Such properties can be determined by methods known in the art, for example, using the computer-modelling program OLIGO^® Primer Analysis Software (distributed by National Biosciences, Inc., Plymouth, MN).

Non-limiting examples of suitable primer sequences include SEQ ID NOs: 18, 19, 24, 25, 44, 45, 66, 67, 72, 73 and 74, shown in Table 1, as well as primers comprising at least 7 consecutive nucleotides of any one of SEQ ID NOs:18, 19, 21, 23, 24, 25, 44, 45, 47, 49, 66, 67, 69, 71, 72, 73 and 74.

As discussed above, the present invention also provides for secondary primers that are capable of amplifying strains from one or more species of Listeria. In one embodiment, the secondary primers are specific to strains of one species of Listeria. In another embodiment, the secondary primers are specific to strains of Listeria grayi. In a further embodiment, the secondary primers comprise at least 7 consecutive nucleotides of the sequence as set forth in any one of SEQ ID NOs:2, 3, 4, 28, 29, 30, 53, 54 or 55, or the complement thereof.

Exemplary secondary primers include those that target the same region of the ssrA gene as the sequences provided above, but which include the specific sequence characteristic of the species that they are targeted against. By way of example, primers targeted to L. grayi can be designed to target the same region of the ssrA gene as SEQ ID NOs: 44 and 45 shown in Table 1. Primers specific for L. grayi, however, could include additional nucleotides and/or a number of substitutions such that they exactly match the L. grayi ssrA gene sequence. Probes

In order to specifically detect various Listeria species, the probe polynucleotides of the invention are designed to correspond to or be complementary to a portion of one of the consensus sequences shown in SEQ ID NOs: 16, 42_. and 64. The probe polynucleotides, therefore, comprise at least 7 consecutive nucleotides of the sequence set forth in any one of SEQ ID NOs: 16, 42 or 64, or the complement thereof. As indicated above, highly conserved regions were identified within the Listeria consensus sequences, hi one embodiment, therefore, the present invention provides for probe polynucleotides comprising at least 7 consecutive nucleotides of the sequence set forth in any one of SEQ ID NO: 17, 43 or 65, or the complement thereof.

Non-limiting examples of suitable probe sequences include SEQ ID NOs: 21, 23, 47, 49, 69 and 71 shown in Table 1, as well as probes comprising at least 7 consecutive nucleotides of any one of SEQ ID NOs:18, 19, 21, 24, 25, 44, 45, 47, 66, 67, 69, 72, 73 and 74, or the complement thereof.

The present invention also contemplates secondary probes that are capable of detecting strains from one or more species of Listeria. In one embodiment, the secondary probes are provided that are specific to strains of one species of Listeria. In another embodiment, the secondary probes are specific to strains of Listeria grayi.

As discussed above with respect to secondary primer sequences, exemplary secondary probes include those that target the same region of the ssrA gene as the probe sequences provided above, but which include the specific sequence characteristic of the species that they are targeted against. By way of example, probes targeted to L. grayi can be designed to target the same region of the ssrA gene as SEQ ID NOs:47 and 49 shown in Table 1. Probes specific for L. grayi, however, could include additional nucleotides and/or a number of substitutions such that they exactly match the L. grayi ssrA gene sequence.

Various types of probes known in the art are contemplated by the present invention. For example, the probe may be a hybridization probe, the binding of which to a target nucleotide sequence can be detected using a general DNA binding dye such as ethidium bromide, SYBR^® Green, SYBR^® Gold and the like. Alternatively, the probe can incorporate one or more detectable labels. Detectable labels are molecules or moieties a property or characteristic of which can be detected directly or indirectly and are chosen such that the ability of the probe to hybridize with its target sequence is not affected. Methods of labelling nucleic acid sequences are well-known in the art (see, for example, Ausubel et al, (1997 & updates) Current Protocols in Molecular Biology, Wiley & Sons, New York).

Labels suitable for use with the probes of the present invention include those that can be directly detected, such as radioisotopes, fluorophores, chemiluminophores, enzymes, colloidal particles, fluorescent microparticles, and the like. One skilled in the art will understand that directly detectable labels may require additional components, such as substrates, triggering reagents, light, and the like to enable detection of the label. The present invention also contemplates the use of labels that are detected indirectly. Indirectly detectable labels are typically specific binding members used in conjunction with a "conjugate" that is attached or coupled to a directly detectable label. Coupling chemistries for synthesising such conjugates are well-known in the art and are designed such that the specific binding property of the specific binding member and the detectable property of the label remain intact. As used herein, "specific binding member" and "conjugate" refer to the two members of a binding pair, i.e. two different molecules, where the specific binding member binds specifically to the probe, and the "conjugate" specifically binds to the specific binding member. Binding between the two members of the pair is typically chemical or physical in nature. Examples of such binding pairs include, but are not limited to, antigens and antibodies; avidin/streptavidin and biotin; haptens and antibodies specific for haptens; complementary nucleotide sequences; enzyme cofactors / substrates and enzymes; and the like.

In one embodiment of the present invention, the probe is labelled with a fluorophore. The probe may additionally incorporate a quencher for the fluorophore. Fluorescently labelled probes can be particularly useful for the real-time detection of target nucleotide sequences in a test sample. Examples of probes that are labelled with both a fluorophore and a quencher that are contemplated by the present invention include, but are not limited to, molecular beacon probes and TaqMan probes. Such probes are well known in the art (see for example, U.S. Patent Nos. 6,150,097; 5,925,517 and 6,103,476; Marras et at, "Genotyping single nucleotide polymorphisms with molecular beacons." In Kwok, P. Y. (ed.), "Single nucleotide polymorphisms: methods and protocols," Vol. 212, pp. 111-128, Humana Press, Totowa, NJ.)

A molecular beacon probe is a hairpin shaped oligonucleotide sequence, which undergoes a conformational change when it hybridizes to a perfectly complementary target sequence. The secondary structure of a typical molecular beacon probe includes a loop sequence, which is capable of hybridizing to a target sequence and a pair of arm sequences. One "arm" of the probe sequence is attached to a fluorophore, while the other "arm" of the probe is attached to a quencher. The arm sequences are complementary to each other and hybridize together to form a molecular duplex such that the molecular beacon adopts a hairpin conformation. In this conformation, the fluorophore and quencher are in close proximity and interact such that emission of fluorescence is prevented. The loop sequence remains un-hybridized. Hybridization between the loop sequence and the target sequence forces the molecular beacon probe to undergo a conformational change in which arm sequences are forced apart and the fluorophore is physically separated from the quencher. As a result, the fluorescence of the fluorophore is restored. The fluorescence generated can be monitored and related to the presence of the target nucleotide sequence. If no target sequence is present in the sample, no fluorescence will be observed. This methodology, as described further below, can also be used to quantify the amount of target nucleotide in a sample. By way of example, Figures 3, 6 and 9 depict the secondary structure of exemplary hairpin loop molecular beacons having sequences corresponding to SEQ ID NO:20, SEQ ID NO:46 and SEQ ID NO:68, respectively.

Wavelength-shifting molecular beacon probes which incorporate two fluorophores, a "harvester fluorophore and an "emitter" fluorophore (see, Kramer, et at, (2000) Nature Biotechnology, 18:1191-1196) are also contemplated. When a wavelength- shifting molecular beacon binds to its target sequence and the hairpin opens, the energy absorbed by the harvester fluorophore is transferred by fluorescence resonance energy transfer (FRET) to the emitter, which then fluoresces. Wavelength-shifting molecular beacons are particularly suited to multiplex assays.

TaqMan^® probes are dual-labelled fluorogenic nucleic acid probes that function on the same principles as molecular beacons. TaqMan^® probes are composed of a polynucleotide that is complementary to a target sequence and is labelled at the 5' terminus with a fluorophore and at the 3' terminus with a quencher. TaqMan^® probes, like molecular beacons, are typically used as real-time probes in amplification reactions, hi the free probe, the close proximity of the fluorophore and the quencher, ensures that the fluorophore is internally quenched. During the extension phase of the amplification reaction, the probe is cleaved by the 5' nuclease activity of the polymerase and the fluorophore is released. The released fluorophore can then fluoresce and produce a detectable signal.

Linear probes comprising a fluorophore and a high efficiency dark quencher, such as the Black Hole Quenchers (BHQ™; Biosearch Technologies, Inc., Novato, CA) are also contemplated. As is known in the art, the high quenching efficiency and lack of native fluorescence of the BHQ™ dyes allows "random-coil" quenching to occur in linear probes labelled at one terminus with a fluorophore and at the other with a BHQ™ dye thus ensuring that the fluorophore does not fluoresce when the probe is in solution. Upon binding its target sequence, the probe stretches out spatially separating the fluorophore and quencher and allowing the fluorophore to fluoresce. One skilled in the art will appreciate that the BHQ™ dyes can also be used as the quencher moiety in molecular beacon or TaqMan^® probes.

As an alternative to including a fluorophore and a quencher in a single molecule, two fluorescently labelled probes that anneal to adjacent regions of the target sequence can be used. One of these probes, a donor probe, is labelled at the 3 ' end with a donor fluorophore, such as fluorescein, and the other probe, the acceptor probe, is labelled at the 5 ' end with an acceptor fluorophore, such as LC Red 640 or LC Red 705. When the donor fluorophore is stimulated by the excitation source, energy is transferred to the acceptor fluorophore by FRET resulting in the emission of a fluorescent signal. In addition to providing primers and probes as separate molecules, the present invention also contemplates polynucleotides that are capable of functioning as both primer and probe in an amplification reaction. Such combined primer/probe polynucleotides are known in the art and include, but are not limited to, Scorpion probes, duplex Scorpion probes, Lux™ primers and Amplifluor™ primers.

Scorpion probes consist of, from the 5 ' to 3 ' end, (i) a fluorophore, (ii) a specific probe sequence that is complementary to a portion of the target sequence and is held in a hairpin configuration by complementary stem loop sequences, (iii) a quencher, (iv) a PCR blocker (such as, hexethylene glycol) and (v) a primer sequence. After extension of the primer sequence in an amplification reaction, the probe folds back on itself so that the specific probe sequence can bind to its complement within the same DNA strand. This opens up the hairpin and the fluorophore can fluoresce. Duplex Scorpion probes are a modification of Scorpion probes in which the fluorophore- coupled probe/primer containing the PCR blocker and the quencher-coupled sequence are provided as separate complementary polynucleotides. When the two polynucleotides are hybridized as a duplex molecule, the fluorophore is quenched. Upon dissociation of the duplex when the primer/probe binds the target sequence, the fluorophore and quencher become spatially separated and the fluorophore fluoresces.

The Amplifluor Universal Detection System also employs fluorophore/quencher combinations and is commercially available from Chemicon International (Temecula, CA).

hi contrast, Lux™ primers incorporate only a fluorophore and adopt a hairpin structure in solution that allows them to self-quench. Opening of the hairpin upon binding to a target sequence allows the fluorophore to fluoresce.

Suitable fluorophores and/or quenchers for use with the polynucleotides of the present invention are known in the art (see for example, Tgayi et ah, Nature BiotechnoL, 16:49-53 (1998); Marras et al, Genet. Anal: Biomolec. Eng., 14:151-156 (1999)). Many fluorophores and quenchers are available commercially, for example from Molecular Probes (Eugene, OR) or Biosearch Technologies, Inc. (Novato, CA). Examples of fluorophores that can be used in the present invention include, but are not limited to, fluorescein and fluorescein derivatives, such as 6-carboxyfluoroscein (FAM), S'-tetrachlorofluorescein phosphoroamidite (TET), tetrachloro-6- carboxyfluoroscein, VIC and JOE, 5-(2'-aminoethyl)aminonaphthalene-l-sulphonic acid (EDANS), coumarin and coumarin derivatives, Lucifer yellow, Texas red, tetramethylrhodamine, 5-carboxyrhodamine, cyanine dyes (such as Cy5) and the like. Pairs of fluorophores suitable for use as FRET pairs include, but are not limited to, fluorescein/rhodamine, fluorescein/Cy5, fluorescein/Cy5.5, fluorescein/LC Red 640, fluorescein/LC Red 750, and phycoerythrin/Cy7. Quenchers include, but are not limited to, 4'-(4-dimethylaminophenylazo)benzoic acid (DABCYL), 4- dimethylaminophenylazophenyl-4 '-maleimide (DABMI), tetramethykhodamine, carboxytetramethylrhodamine (TAMRA), BHQ™ dyes and the like.

Methods of selecting appropriate sequences for and preparing the various primers and probes are known in the art. For example, the polynucleotides can be prepared using conventional solid-phase synthesis using commercially available equipment, such as that available from Applied Biosystems USA Inc. (Foster City, California), DuPont, (Wilmington, Del.), or Milligen (Bedford, Mass.). Methods of coupling fluorophores and quenchers to nucleic acids are also in the art.

In one embodiment of the present invention, the probe polynucleotide is a molecular beacon. In general, in order to form a hairpin structure effectively, molecular beacons are at least 17 nucleotides in length. In accordance with this aspect of the invention, therefore, the molecular beacon probe is typically between about 17 and about 40 nucleotides in length. Within the probe, the loop sequence that corresponds to or is complementary to the target sequence typically is about 7 to about 32 nucleotides in length, while the stem (or "arm") sequences are each between about 4 and about 9 nucleotides in length. As indicated above, part of the stem sequences of a molecular beacon may also be complementary to the target sequence, hi one embodiment of the present invention, the loop sequence of the molecular beacon is between about 10 and about 30 nucleotides in length. In other embodiments, the loop sequence of the molecular beacon is between about 15 and about 30 nucleotides in length. In accordance with the present invention, the loop region of the molecular beacon probe comprises at least 7 consecutive nucleotides of the sequence as set forth in SEQ ID NO: 16, 42 or 64, or the complement thereof. In a specific embodiment, the loop region of the molecular beacon probe comprises at least 7 consecutive nucleotides of the sequence as set forth in any one of SEQ ID NOs :21, 23, 47, 49, 69 and 71.

AMPLIFICATION AND DETECTION

In accordance with the present invention, detection of Listeria species involves subjecting a test sample to an amplification reaction in order to obtain an amplification product, or amplicon comprising the target nucleotide sequence, which in turn comprises all or a portion of one or more consensus sequences.

As used herein, an "amplification reaction" refers to a process that increases the number of copies of a particular nucleic acid sequence by enzymatic means. Amplification procedures are well-known in the art and include, but are not limited to, polymerase chain reaction (PCR), TMA, rolling circle amplification, nucleic acid sequence based amplification (NASBA), strand displacement amplification (SDA) and Q-beta replicase amplification. One skilled in the art will understand that for use in certain amplification techniques the primers described above may need to be modified, for example, SDA primers comprise additional nucleotides near the 5' end that constitute a recognition site for a restriction endonuclease. Similarly, NASBA primers comprise additional nucleotides near the 5' end that are not complementary to the target sequence but which constitute an RNA polymerase promoter. Polynucleotides thus modified are considered to be within the scope of the present invention.

In one embodiment of the present invention, the target sequence is amplified by PCR. PCR is a method, known in the art for amplifying a nucleotide sequence using a heat stable polymerase and a pair of primers, one primer (the forward primer) complementary to the (+)-strand at one end of the sequence to be amplified and the other primer (the reverse primer) complementary to the (-)- strand at the other end of the sequence to be amplified. Newly synthesized DNA strands can subsequently serve as templates for the same primer sequences and successive rounds of strand denaturation, primer annealing, and strand elongation, produce rapid and highly specific amplification of the target sequence. PCR can thus be used to detect the existence of a defined sequence in a DNA sample. The term "PCR" as used herein refers to the various forms of PCR known in the art including, but not limited to, quantitative PCR, reverse-transcriptase PCR, real-time PCR, hot start PCR, long PCR, LAPCR, multiplex PCR, touchdown PCR, and the like. "Real-time PCR" refers to a PCR reaction in which the amplification of a target sequence is monitored in real time by, for example, the detection of fluorescence emitted by the binding of a labelled probe to the amplified target sequence.

Thus, in one embodiment, the present invention provides for a method of amplifying a Listeria ssrA target nucleotide sequence of less than about 500 nucleotides in length and comprising at least 50 consecutive nucleotides of the sequence set forth in any one of SEQ ID NOs: 16, 42 or 64 using a pair of polynucleotide primers, each member of the primer pair comprising at least 7 nucleotides of the sequence as set forth in SEQ ID NO:1, or the complement thereof. In another embodiment, the present invention provides for a method of amplifying a Listeria ssrA target nucleotide sequence of less than about 500 nucleotides in length selected from the group of: (i) a target sequence comprising at least 50 consecutive nucleotides of the sequences set forth in SEQ ID NO: 16 and in SEQ. ID NO:64; (ii) a target sequence comprising at least 50 consecutive nucleotides of the sequences set forth in SEQ ID NO: 64 and in SEQ ID NO:42, and (iii) a target sequence comprising at least 50 consecutive nucleotides of the sequences set forth in SEQ ID NO: 16 and in SEQ ID NO:42, using a pair of polynucleotide primers, each member of the primer pair comprising at least 7 nucleotides of the sequence as set forth in SEQ ID NO:1, or the complement thereof.

As discussed above, the present invention also contemplates a system for detection of two Listeria target sequences. Accordingly, in an alternative embodiment, the invention provides for a method of concurrently amplifying two Listeria ssrA target nucleotide sequences of less than about 500 nucleotides in length, the first target sequence comprising at least 50 consecutive nucleotides of the sequence as set forth in SEQ ID NO: 16, 42 or 64 and the second target nucleotide sequence comprising at least 50 consecutive nucleotides of the sequence as set forth in SEQ ID NO: 16, 42 or 64, using two pairs of polynucleotide primers, each member of each pair comprising at least 7 nucleotides of the sequence as set forth in SEQ ID NO:1, or the complement thereof. In a further embodiment, one pair of primers is a pair of primary primers and the other is a pair of secondary primers, wherein the secondary pair of primers is capable of amplifying a target sequence from a species of Listeria that is not amplified by the primary pair of primers, or that is amplified with low efficiency by the primary pair of primers. The target sequence amplified by the primary and secondary primers can thus be the same or different. The product of the amplification reaction can be detected by a number of means known to individuals skilled in the art. Examples of such detection means include, for example, gel electrophoresis and/or the use of polynucleotide probes. In one embodiment of the invention, the amplification products are detected through the use of polynucleotide probes. Such polynucleotide probes are described in detail above.

One embodiment of the invention, therefore, provides for a method of amplifying and detecting & Listeria ssrA target nucleotide sequence of less than about 500 nucleotides in length and comprising at least 50 consecutive nucleotides of the sequence set forth in SEQ ID NO: 16 using a combination of polynucleotides, the combination comprising a pair of polynucleotide primers comprising at least 7 nucleotides of the sequence as set forth in SEQ ID NO:1, or the complement thereof, and a polynucleotide probe comprising at least 7 consecutive nucleotides of the sequence as set forth in SEQ ID NO: 16, or the complement thereof.

Another embodiment of the invention provides for a method of amplifying and detecting & Listeria ssrA target nucleotide sequence of less than about 500 nucleotides in length and comprising at least 50 consecutive nucleotides of the sequence set forth in SEQ ID NO:42 using a combination of polynucleotides, the combination comprising a pair of polynucleotide primers comprising at least 7 nucleotides of the sequence as set forth in SEQ ID NO:1, or the complement thereof, and a polynucleotide probe comprising at least 7 consecutive nucleotides of the sequence as set forth in SEQ ID NO:42, or the complement thereof. Another embodiment of the invention provides for a method of amplifying and detecting a. Listeria ssrA target nucleotide sequence of less than about 500 nucleotides in length and comprising at least 50 consecutive nucleotides of the sequence set forth in SEQ ID NO:64 using a combination of polynucleotides, the combination comprising a pair of polynucleotide primers comprising at least 7 nucleotides of the sequence as set forth in SEQ ID NO:1, or the complement thereof, and a polynucleotide probe comprising at least 7 consecutive nucleotides of the sequence as set forth in SEQ ID NO:64, or the complement thereof.

The system of the present invention also provides for a method of amplifying and detecting a Listeria ssrA target nucleotide sequence comprising two or more consensus sequences. Accordingly, a further embodiment provides for a method of amplifying and detecting & Listeria ssrA target nucleotide sequence of less than about 500 nucleotides in length and comprising at least 50 consecutive nucleotides two or more of the consensus sequences as set forth in SEQ ID NOs: 16, 42 and 64 using a combination of polynucleotides, the combination comprising one or more polynucleotide primers comprising at least 7 nucleotides of the sequence as set forth in SEQ ID NO:1, or the complement thereof, and one or more polynucleotide probes comprising at least 7 consecutive nucleotides of the sequence as set forth in SEQ ID NO: 16, 42 or 64, or the complement thereof.

A further embodiment of the present invention provides for a method of amplifying and detecting two Listeria ssrA target nucleotide sequences concurrently using a combination of polynucleotides, the combination comprising at least two pairs of polynucleotide primers, each primer comprising at least 7 nucleotides of the sequence as set forth in SEQ ID NO:1, or the complement thereof, wherein each set of primers is capable of amplifying a Listeria ssrA target nucleotide sequence comprising at least 50 consecutive nucleotides of any one of SEQ ID NOs: 16, 42 or 64 and at least two corresponding polynucleotide probes, i.e. probes comprising at least 7 consecutive nucleotides of the sequence as set forth in any one of SEQ ID NOs: 16, 42 or 64, or the complement thereof. The Listeria ssrA target nucleotide sequences can be the same sequence from different Listeria species, or they can be different target nucleotide sequences. For example, one pair of primers and one probe can be a primary set of polynucleotides, as described above, and the second pair of primers and probe can be a secondary set of polynucleotides designed to amplify and detect one or more species of Listeria that are not efficiently amplified and/or detected by the primary set of polynucleotides.

It will be readily appreciated that a procedure that allows both amplification and detection of target Listeria nucleic acid sequences to take place concurrently in a single unopened reaction vessel would be advantageous. Such a procedure would avoid the risk of "carry-over" contamination in the post-amplification processing steps, and would also facilitate high-throughput screening or assays and the adaptation of the procedure to automation. Furthermore, this type of procedure allows "real time" monitoring of the amplification reaction, as discussed above, as well as conventional "end-point" monitoring. In one embodiment, the detection is accomplished in real time in order to facilitate rapid detection. In a specific embodiment, detection is accomplished in real time through the use of one or more molecular beacon probe.

The present invention thus provides for methods to specifically amplify and detect Listeria target nucleotide sequences in a test sample in a single tube format using the polynucleotide primers, and optionally one or more probes, described herein. Such methods may employ dyes, such as SYBR^® Green or SYBR^® Gold that bind to the amplified target sequence, or an antibody that specifically detects the amplified target sequence. The dye or antibody is included in the reaction vessel and detects the amplified sequences as it is formed. Alternatively, one or more labelled polynucleotide probe (such as a molecular beacon or TaqMan® probe) distinct from the primer sequences, which is complementary to a region of the amplified sequence, may be included in the reaction, or one of the primers may act as a combined primer/probe, such as a Scorpion probe. Such options are discussed in detail above.

Thus, a general method of detecting one or more Listeria species in a sample is provided that comprises contacting a test sample suspected of containing, or known to contain, one or more Listeria ssrA target nucleotide sequences with a combination of polynucleotides comprising at least one polynucleotide primer and at least one polynucleotide probe or primer/probe, as described above, under conditions that permit amplification of said one or more target sequences, and detecting any amplified target sequence as an indication of the presence of one or more Listeria species in the sample. A "test sample" as used herein is a biological sample suspected of containing, or known to contain, one or more Listeria target nucleotide sequences.

In one embodiment of the present invention, a method using the polynucleotide primers and probes or primer/probes is provided to specifically amplify and detect one or more Listeria target nucleotide sequences in a test sample, the method generally comprising the steps of:

(a) forming a reaction mixture comprising a test sample, amplification reagents, one or more polynucleotide probe sequence capable of specifically hybridising to a portion of a Listeria target nucleotide sequence and one or more polynucleotide primer corresponding to or complementary to a Listeria ssrA gene comprising said one or more target nucleotide sequences;

(b) subjecting the mixture to amplification conditions to generate at least one copy of the one or more target nucleotide sequences, or a nucleic acid sequence complementary thereto, thereby producing amplified target nucleotide sequences;

(c) hybridizing the probe to the amplified target nucleotide sequences, so as to form probe:target hybrids; and

(d) detecting the probe:target hybrids as an indication of the presence of the one or more Listeria ssrA target nucleotide sequences in the test sample.

When more than one probe is employed in a method of the invention, the probes can be capable of specifically hybridising to the same Listeria ssrA target nucleotide sequence when one target nucleotide sequence is being detected, or to different target nucleotide sequences when more than one target nucleotide sequences are being detected, or to two different consensus sequences within a single target nucleotide sequence. In another embodiment of the present invention, the method employs one or more labelled probe in step (a). In a specific embodiment of the present invention, the amplification and detection steps take place concurrently and the method thus provides for "real time" detection of Listeria species in the test sample.

The term "amplification reagents" includes conventional reagents employed in amplification reactions and includes, but is not limited to, one or more enzymes having nucleic acid polymerase activity, enzyme cofactors (such as magnesium or nicotinamide adenine dinucleotide (NAD)), salts, buffers, nucleotides such as deoxynucleotide triphosphates (dNTPs; for example, deoxyadenosine triphosphate, deoxyguanosine triphosphate, deoxycytidine triphosphate and deoxythymidine triphosphate) and other reagents that modulate the activity of the polymerase enzyme or the specificity of the primers.

It will be readily understood by one skilled in the art that step (b) of the above method can be repeated several times prior to step (c) by thermal cycling the reaction mixture by techniques known in the art and that steps (b), (c) and (d) may take place concurrently such that the detection of the amplified sequence takes place in real time. In addition, variations of the above method can be made depending on the intended application of the method, for example, the polynucleotide probe may be a combined primer/probe, or it may be a separate polynucleotide probe. Additional steps may be incorporated before, between or after those listed above as necessary, for example, the test sample may undergo enrichment, extraction and/or purification steps to isolate nucleic acids therefrom prior to the amplification reaction, and/or the amplified product may be submitted to purification/isolation steps or further amplification prior to detection, and/or the results from the detection step (d) may be analysed in order to quantify the amount of target present in the sample or to compare the results with those from other samples. These and other variations will be apparent to one skilled in the art and are considered to be within the scope of the present invention.

hi one embodiment of the present invention, the method is a real-time PCR assay utilising two polynucleotide primers and a molecular beacon probe. In a further embodiment, the real-time PCR assay employs a combination of two pairs of polynucleotide primers and two molecular beacon probes for amplification and detection of two different ssrA target nucleotide sequences. In a further embodiment of the invention, the real-time PCR assay employs a combination of two pairs of polynucleotide primers and two molecular beacon probes for amplification and detection of the same ssrA target nucleotide sequence. In another embodiment, the real-time PCR assay employs a combination of one or more pairs of polynucleotide primers and two molecular beacon probes for amplification and detection of the same ssrA target nucleotide sequence, with one molecular beacon probe being designed to detect one species of Listeria only.

DIAGNOSTIC ASSAYS TO DETECT LISTERIA SPECIES

The present invention provides for diagnostic assays using the polynucleotide primers and/or probes that can be used for highly specific detection of Listeria spp. in a test sample. In accordance with the present invention, the diagnostic assays are capable of detecting at least five of the six known Listeria species. In one embodiment, the diagnostic assays are capable of detecting all six known Listeria species. The diagnostic assays comprise amplification and detection of Listeria target nucleotide sequence(s) as described above. The diagnostic assays can be qualitative or quantitative and can involve real-time monitoring of the amplification reaction or conventional end-point monitoring.

In one embodiment, the invention provides for diagnostic assays that do not require post-amplification manipulations and thereby minimise the amount of time required to conduct the assay. For example, in a specific embodiment, there is provided a diagnostic assay, utilising the primers and probes described herein, that can be completed using real time PCR technology in about 54 hours and generally less that 24 hours.

Such diagnostic assays are particularly useful for detection of contamination of various foodstuffs by one or more species of Listeria. Thus, in one embodiment, the present invention provides a rapid and sensitive diagnostic assay for the detection of Listeria contamination of a food sample. Foods that can be analysed using the diagnostic assays include, but are not limited to, dairy products such as milk, including raw milk, cheese, yoghurt, ice cream and cream; raw, cooked and cured meats and meat products, such as beef, pork, lamb, mutton, poultry (including turkey, chicken), game (including rabbit, grouse, pheasant, duck), minced and ground meat (including ground beef, ground turkey, ground chicken, ground pork); eggs; fruits and vegetables; nuts and nut products, such as nut butters; seafood products including fish and shellfish; and fruit or vegetable juices. The diagnostic assays may also be used to detect Listeria contamination of drinking water.

While the primary focus of Listeria detection is food products, the present invention also contemplates the use of the primers and probes in diagnostic assays for the detection of Listeria contamination of other biological samples, such as patient specimens in a clinical setting, for example, faeces, blood, saliva, throat swabs, urine, mucous, and the like, as well as Listeria contamination of surfaces and instruments, such as surgical or dental instruments. The diagnostic assays are also useful in the assessment of microbiologically pure cultures, and in environmental and pharmaceutical quality control processes.

The test sample can be used in the assay either directly {i.e. as obtained from the source) or following one or more pre-treatment steps to modify the character of the sample. Thus, the test sample can be pre-treated prior to use, for example, by disrupting cells or tissue, extracting the microbial content from the sample (such as a swab or wipe test sample), enhancing the microbial content of the sample by culturing in a suitable medium, preparing liquids from solid materials, diluting viscous fluids, filtering liquids, distilling liquids, concentrating liquids, inactivating interfering components, adding reagents, isolating and/or purifying nucleic acids, and the like. In one embodiment of the present invention, the test sample is subjected to one or more steps to isolate, or partially isolate, nucleic acids therefrom.

As indicated above, the polynucleotide primers and probes of the invention can be used in assays to quantitate the amount of Listeria target nucleotide sequence(s) in a test sample. Thus, the present invention provides for a method to specifically amplify, detect and quantitate one or more Listeria target nucleotide sequences in a test sample, the methods generally comprising the steps of: (a) forming a reaction mixture comprising a test sample, amplification reagents, one or more polynucleotide probe capable of specifically hybridising to a portion of a Listeria target nucleotide sequence and one or more polynucleotide primer corresponding to or complementary to a Listeria ssrA gene comprising said one or more target nucleotide sequences;

(c) hybridizing the probe to the amplified target nucleotide sequences, so as to form probe:target hybrids;

(d) detecting the probe:target hybrids; and

(e) analysing the amount of probe:target hybrid present as an indication of the amount of target nucleotide sequence(s) present in the test sample.

The steps of this method may also be varied as described above for the amplification/detection method.

In one embodiment, the method employs one or more labelled polynucleotide probe in step (a) and steps (d) and (e) are as follows:

(d) detecting the probe:target hybrid by detecting the signal produced by the hybridized labelled probe; and

(e) analysing the amount of signal produced as an indication of the amount of target nucleotide sequence present in the test sample..

Step (e) can be conducted, for example, by comparing the amount of probe:target hybrid present to a standard or utilising one of a number of statistical methods known in the art that do not require a standard.

Various types of standards for quantitative assays are known in the art. For example, the standard can consist of a standard curve compiled by amplification and detection of known quantities of a Listeria target nucleotide sequence under the assay conditions. Alternatively, relative quantitation can be performed without the need for a standard curve (see, for example, Pfaffl, MW. (2001) Nucleic Acids Research 29(9):2002-2007). In this method, a reference gene is selected against which the expression of the target gene can be compared and an additional pair of primers and an appropriate probe are included in the reaction in order to amplify and detect a portion of the selected reference gene. The reference gene is usually a gene that is expressed constitutively, for example, a house-keeping gene.

Another similar method of quantification is based on the inclusion of an internal standard in the reaction. Such internal standards generally comprise a control target nucleotide sequence and a control polynucleotide probe. The internal standard can further include an additional pair of primers that specifically amplify the control target nucleotide sequence and are unrelated to the polynucleotides of the present invention. Alternatively, the control target sequence can contain primer target sequences that allow specific binding of the assay primers but a different probe target sequence. This allows both the Listeria target sequence and the control sequence to be amplified with the same primers, but the amplicons are detected with separate probe polynucleotides. Typically, when a reference gene or an internal standard is employed, the reference/control probe incorporates a detectable label that is distinct from the label incorporated into the Listeria target sequence specific probe(s). The signals generated by these two labels when they bind their respective target sequences can thus be distinguished.

hi the context of the present invention, a control target nucleotide sequence is a nucleic acid sequence that (i) can be amplified either by the Listeria target sequence specific primers employed in the assay or by control primers, (ii) specifically hybridizes to the control probe under the assay conditions and (iii) does not exhibit significant hybridization to the Listeria target sequence specific probe(s) under the same conditions. One skilled in the art will recognise that the actual nucleic acid sequences of the control target nucleotide and the control probe are not important provided that they both meet the criteria outlined above. The diagnostic assays can be readily adapted for high-throughput. High-throughput assays provide the advantage of processing many samples simultaneously and significantly decrease the time required to screen a large number of samples. The present invention, therefore, contemplates the use of the polynucleotides of the present invention in high-throughput screening or assays to detect and/or quantitate Listeria target nucleotide sequences in a plurality of test samples.

For high-throughput assays, reaction components are usually housed in a multi- container carrier or platform, such as a multi-well microtitre plate, which allows a plurality of assays each containing a different test sample to be monitored simultaneously. Control samples can also be included in the plates to provide internal controls for each plate. Many automated systems are now available commercially for high-throughput assays, as are automation capabilities for procedures such as sample and reagent pipetting, liquid dispensing, timed incubations, formatting samples into microarrays, microplate thermocycling and microplate readings in an appropriate detector, resulting in much faster throughput times.

KITS AND PACKAGES FOR THE DETECTION OF LISTERIA SPECIES

The present invention further provides for kits for detecting Listeria spp. in a variety of samples. In general, the kits comprise one or more pairs of primers and one or more probes capable of amplifying and detecting one or more Listeria target nucleotide sequences as described above. One of the primers and the probe(s) may be provided in the form of a single polynucleotide, such as a Scorpion probe, as described above. The probe(s) provided in the kit can be unlabelled, or can incorporate a detectable label, such as a fluorophore or a fluorophore and a quencher, or the kit may include reagents for labelling the probe(s). The primers/probes can be provided in separate containers or in an array format, for example, pre-dispensed into microtitre plates.

One embodiment of the present invention provides for kits comprising a combination of primers and probes that are capable of amplifying and detecting a single Listeria target nucleotide sequence. Another embodiment provides for kits comprising a combination of primers and probes that are capable of amplifying and detecting different Listeria ssrA target nucleotide sequences. Another embodiment provides for kits comprising a combination of primers and probes that are capable of amplifying and detecting the same ssrA target nucleotide sequence from different Listeria species.

The kits can optionally include amplification reagents, such as buffers, salts, enzymes, enzyme co-factors, nucleotides and the like. Other components, such as buffers and solutions for the enrichment, isolation and/or lysis of bacteria in a test sample, extraction of nucleic acids, purification of nucleic acids and the like may also be included in the kit. One or more of the components of the kit may be lyophilised and the kit may further comprise reagents suitable for the reconstitution of the lyophilised components.

The various components of the kit are provided in suitable containers. As indicated above, one or more of the containers may be a microtitre plate. Where appropriate, the kit may also optionally contain reaction vessels, mixing vessels and other components that facilitate the preparation of reagents or nucleic acids from the test sample.

The kit may additionally include one or more controls. For example, control polynucleotides (primers, probes, target sequences or a combination thereof) may be provided that allow for quality control of the amplification reaction and/or sample preparation, or that allow for the quantitation of Listeria target nucleotide sequences.

The kit can additionally contain instructions for use, which may be provided in paper form or in computer-readable form, such as a disc, CD, DVD or the like.

The present invention further contemplates that the kits described above may be provided as part of a package that includes computer software to analyse data generated from the use of the kit.

The invention will now be described with reference to specific examples. It will be understood that the following examples are intended to describe embodiments of the invention and are not intended to limit the invention in any way. EXAMPLES

Example 1: Determination of a First Consensus Sequence in Listeria Species ssrA Gene Sequences

The coding region of the ssrA gene from 14 different Listeria isolates were sequenced and aligned using the multiple alignment program Clustal W™. The resulting alignment was used to identify short DNA regions that were conserved within the Listeria group, yet which are excluded from other bacteria. Figure 1 depicts a sample of such an alignment in which a portion of the coding strand of the ssrA gene of 14 different Listeria isolates has been aligned.

A 87 nucleotide conserved sequence was identified as described above (SEQ ID NO: 16).

AACGTCAAAGCCAATAATAACTGGCAAAGAAAAACAAAACCTAGCTTTCG CTGCCTAATAAGCAGTAGCATAGCTGATCCTCCGTGC [SEQ ID NO:16]

This unique and conserved region of the ssrA Listeria gene sequences designated consensus sequence #1 and was used to design highly specific primers for the PCR amplification of this region of the ssrA gene.

Example 2: Generation of DNA Primers for Amplification of ssrA Consensus Sequence #1

Within consensus sequence #1 two regions that could serve as primer target sequences were identified. These primer target sequences were used to design a pair of primers to allow efficient PCR amplification of this sequence. The primer sequences are shown below:

Forward primer #1: 5 '-AACGTCAAAGCCAATAATAACTG-3 ' [SEQ ID NO: 18]

Reverse primer #1: 5'-GCACGGAGGATCAGCTAT-S' [SEQ ID NO:19]

In the alignment presented in Figure 1, the positions of forward primer #1 and reverse primer #1 are represented by shaded boxes. Forward primer #1 starts at position 31 and ends at position 53 of the alignment. Reverse primer #1 represents the reverse complement of the region starting at position 100 and ending at position 117.

The following pair of primers can also be used to amplify consensus sequence #1:

Forward primer #2: 5'-ACGTCAAAGCCAATAATAACTGGC-S' [SEQ ID NO:24]

Reverse primer #2: 5'-GATGCACGGAGGATCAGCTAT-S' [SEQ ID NO: 25]

Example 3; Generation of Molecular Beacon Probes Specific for ssrA Consensus Sequence #1

In order to design molecular beacon probes specific for Listeria, a region within consensus sequence #1 was identified which not only was highly conserved in all Listeria isolates but was also exclusive to Listeria isolates. This sequence consisted of a 19 nucleotide region that would be suitable for use as a molecular beacon target sequence. The sequence is provided below:

5'-TAGCTTTCGCTGCCTAATA-S ' [SEQ ID NO:21]

The complement of this sequence is also suitable for use as a molecular beacon target sequence [SEQ ID NO:23].

₅,__{TATTAGGCAGCGAAAGCTA}_₃, __SEQ ^ _NO:23-]

A molecular beacon probe having the sequence shown below was synthesized by Integrated DNA Technologies Lie.

Molecular beacon probe #1 :

5 '-CGCGTAGCTTTCGCTGCCTAATACGCG-S ' [SEQ ID NO:20]

The complement of this sequence (SEQ ID NO:22, shown below) can also be used as a molecular beacon probe for the detecting Listeria.

5'-CGCGTATTAGGCAGCGAAAGCTACGCG-S' [SEQ ID NO:22] The starting material for the synthesis of the molecular beacons was an oligonucleotide that contains a sulfhydryl group at its 5' end and a primary amino group at its 3' end. DABCYL was coupled to the primary amino group utilizing an amine-reactive derivative of DABCYL. The oligonucleotides that were coupled to DABCYL were then purified. The protective trityl moiety was then removed from the 5'-sulfhydryl group and a fluorophore was introduced in its place using an iodoacetamide derivative.

An individual skilled in the art would recognize that a variety of methodologies could be used for synthesis of the molecular beacons. For example, a controlled-pore glass column that introduces a DABCYL moiety at the 3' end of an oligonucleotide has recently become available, which enables the synthesis of a molecular beacon completely on a DNA synthesizer.

Table 2 provides a general overview of the characteristics of molecular beacon probe #1. The beacon sequence shown in Table 2 indicates the stem region in lower case and the loop region in upper case.

Table 2. Description of Molecular Beacon Probe #1.

Beacon sequence (5'-> 3') : cgcgTAGCTTTCGCTGCCTAATAcgcg

Fluorophore (5') : FAM

Quencher (3') : DABCYL

Table 3 provides an overview of the thermodynamics of the folding of molecular beacon probe #1. Calculations were made using MFOLD™ software, or the Oligo Analyzer software package available on Integrated DNA Technologies Inc. web site. Figure 2 shows the arrangement of the PCR primers and molecular beacon probe #1 on the ssrA consensus sequence #1. Numbers in parentheses indicate the positions of the first and last nucleotides of each feature on the PCR product generated with the forward primer #1 and reverse primer #1.

Table 3. Thermodynamics of Molecular Beacon Probe #1.

A further ssrA specific molecular beacon suitable for the detection of Listeria was also prepared as described above. The sequence is shown below (nucleotides in lower case represent the nucleotides that make up the stem of the beacon):

ssrA molecular beacon probe #2 :

5'- cccgT AGCTTTCGCTGCCTAATAcggg -3' [SEQ ID NO:26]

The complement of this sequence (SEQ ID NO:27) can also be used as molecular beacon probes for the detection of Listeria.

5 '- cccgT ATTAGGCAGCGAAAGCTAcggg -3 ' [SEQ ID NO:27]

Example 4; Isolation of DNA from Samples

The following protocol was utilized in order to isolate DNA sequences from samples.

Material needed for DNA extraction:

-Tungsten carbide beads: Qiagen

-Reagent DX: Qiagen

-DNeasy Plant Mini Kit: Qiagen

-Tissue Disruption equipment: Mixer Mill™ 300 (Qiagen)

The following method was followed:

1) Add to a 2 ml screw top tube: 1 tungsten carbide bead and 0.1 g glass beads 212 to 300 μm in width + sample to be analysed + 500 μL of API buffer + 1 μL of Reagent DX + 1 μL of RNase A (100 mg/niL). Extraction control was performed without adding sample to be analysed. 2) heat in Dry-Bath at 8O⁰C for 10 min.

3) mix in a Mixer Mill 300 (MM300) at frequency of 30 Hz [1/s], 2 min.

4) rotate tubes and let stand for 5 min at room temperature.

5) mix in a Mixer Mill 300, frequency 30 Hz, 1 min.

6) place tubes in boiling water for 5 min.

7) centrifuge with a quick spin.

8) add 150 μL of AP2 buffer.

9) mix at frequency of 30 Hz for 30 sec. Rotate tubes and repeat.

10) centrifuge at 13,000 rpm for 1 min.

11) transfer supernatant in to a 2 mL screw top tube containing 800 μL of AP3/E buffer.

12) mix by inverting, centrifuge with a quick spin.

13) add 700 μL of mixture from step 12 to a DNeasy binding column and centrifuge at 800 rpm for 1 minute. Discard eluted buffer. Repeat process with leftover mixture from step 12.

14) add 500 μL of wash buffer (AW buffer) to binding columns and centrifuge for 1 minute at 800 rpm. Discard eluted buffer.

15) add 500 μL of wash buffer (AW buffer) to binding columns and centrifuge for 1 minute at 800 rpm. Discard eluted buffer.

16) centrifuge column again at 8000 rpm for 1 min.

17) place column in a sterile 2 mL tube and add 100 μL of AE elution buffer preheated at 8O⁰C.

18) incubate for 1 min. Centrifuge at max speed for 2 min. Elute twice with 100 μL.

19) keep eluate for PCR amplification.

Time of manipulation: 3 hours. Proceed to prepare PCR reaction for real-time detection. Example 5: Amplification of ssrA Consensus Sequence #1 in Real Time

The effectiveness of forward primer #1 and reverse primer #1 for amplification of Listeria isolates was demonstrated as described generally below.

Genomic DNA from the species and strains presented in Tables 6 and 7 below was isolated as described in Example 4. PCR amplification was undertaken using the conditions described in Tables 4 and 5 below. Amplicons were detected with SYBR^® Green. The intensity of fluorescence emitted by the SYBR Green dye was detected at the elongation stage of each amplification cycle. In Table 4, note that the Qiagen SyBrGreen buffer contains dNTPs and Taq polymerase and 0.125 mM magnesium chloride (final concentration). Inclusion of additional magnesium chloride brings the final concentration to 1.5 mM in the reaction mixture.

Table 4. SyBR Green Reaction mix

Table 5 presents an overview of the cycles used for each step of the PCR amplification.

Table 5. PCR Program

Fluorescence was detected in real-time using a fluorescence monitoring real-time PCR instrument, for example, a BioRad iCycler iQ™ or MJ Research Opticon™. Other instruments with similar fluorescent reading abilities can also be used.

AU 62 strains of Listeria tested (as shown in Table 6) were amplified with forward primer #1 and reverse primer #1. No amplification products were observed with the 242 non-Listeria strains tested (see Table 7). In Tables 6 and 7, the figures in parentheses indicate the number of strains of each species that were tested (if more than one). None of the tested strains provided a positive result.

Table 6. Strains of Listeria used for Positive Validation

Table 7. Strains used for Negative Validation

Organism Serovars Organism Serovars

Acinetobacter calcoaceticus (2) Kurthia zopfii

Λcinetobacter iwoffi Lactobacillus acidophilus

Acinetobacter junii Lactobacillus casei (2)

Aeromonas hydrophila (2) Lactobacillus delbreuckii (2)

Aeromonas salmonicida (2) Lactobacillus helveticus

Alcaligenes faecalis Lactobacillus pentosus

Bacillus amyloliquefaciens (2) Lactobacillus plantarum (2) Organism Serovars Organism Serovars

Bacillus cereus (2) Lactobacillus rhamnosus

Bacillus circulans (2) Lactococcus lactis (2)

Bacillus coagulans (2) Lactococcus raffinolactis

Bacillus firmus Legionella pneumophila (2)

Bacillus lentus Micrococcus luteus (2)

Bacillus licheniformis (2) Moraxella spp.

Bacillus megaterium (2) Mycobacterium smegmatis

Bacillus mycoides Neisseria gonorrhoeae

Bacillus pumilus (2) Neisseria lactamica

Bacillus sphaericus Neisseria meningitidis (2)

Bacillus stearothermophilus Neisseria sica

Bacillus subtilis (2) Nocardia asteroides

Bacillus thuringiensis (2) Pediococcus acidilactici (2)

Bacteroides fragilis Pediococcus pentosaceus

Bifidobacterium adolescentis Proteus mirabilis (2)

Bifidobacterium animalis Proteus penneri (2)

Bifidobacterium bifidum Proteus vulgaris (2)

Bifidobacterium longum Pseudomonas aeruginosa (2)

Bifidobacterium pseudolongum Pseudomonas sp.

Bifidobacterium sp. (2) Pseudomonas mendocina

Bifidobacterium suis Pseudomonas pseudoalcaligenes

Bifidobacterium thermophilus Pseudomonas putida (2)

Bordetella bronchiseptica Pseudomonas stutzeri

Bordetella pertussis Salmonella agona

Borrelia burgdorferi Salmonella arizonae (2) Organism Serovars Organism Serovars

Branhamella catarrhalis Salmonella bongori

Brevibacillus laterosporus Salmonella brandenburg

Campylobacter coli Salmonella choleraesuis (2)

Campylobacter jejuni (2) Salmonella diarizonae

Campylobacter lari (2) Salmonella dublin (2)

Campylobacter rectus Salmonella enteritidis (2)

Cellilomonea sp. Salmonella heidelberg (2)

Chromobacterium violaceum Salmonella houtenae

Chryseobacterium sp. Salmonella indica

Chryseomonas luteola Salmonella infantis (2)

Citrobacter amalonaticus (2) Salmonella montevideo (2)

Citrobacter diversus Salmonella newport (2)

Citrobacter freundii (2) Salmonella paratyphi (4)

Citrobacter koseri Salmonella saintpaul (2)

Citrobacter werhnanii Salmonella senftenberg

Clostridium botulinum (2) Salmonella Stanley

Clostridium butyricum Salmonella thompson (2)

Clostridium difficile Salmonella typhi (2)

Clostridium perfringens (2) Salmonella typhimurium (2)

Clostridium sporogenes Salmonella typhisuis (2)

Clostridium tetani Serratia liquefaciens (2)

Clostridium tyrobutyricum Serratia marcescens (2)

Corynebacterium xerosis_^ Serratia odorifera

Edwardsiella tarda Shigella boydii

Enterobacter aerogenes (2) Shigella dysenteriae (2) Organism Serovars Organism Serovars

Enterobacter amnigenus Shigella flexneri (2)

Enterobacter cloacae (2) Shigella sonnei (2)

Enterobacter intermedius (2) Staphylococcus aureus (2)

Enterobacter taylorae Staphylococcus chromogenes

Enterococcus faecalis (2) Staphylococcus epidermidis (2)

Enterococcus faeciunϊ Staphylococcus intermedius

Enterococcus hirae (2) Staphylococcus lentis

Erwinia herbicola Staphylococcus ludgdunensis

Escherichia blattae (2) Staphylococcus schieiferi

Escherichia coli (4) Staphylococcus xylosus

Escherichia fergusonii Stenotrophomonas maltophilia

Escherichia hermannii (2) Streptococcus agalactiae (2)

Escherichia vulneris Streptococcus bovis

Enterobacter aerogenes Streptococcus pneumoniae (2)

Haemophilus equigenitalis Streptococcus pyogenes (2)

Haemophilus influenzae (2) Streptococcus salivarius

Haemophilus paragallinarum Streptococcus thermophilus

Hafnia alvei (2) Vibrio alginolyticus

Helicobacter pylori Vibrio cholerae (2)

Klebsiella ornithinolytica Vibrio eltor

Klebsiella oxytoca (2) Vibrio fluvialis

Klebsiella planticola (2) Vibrio hollisae

Klebsiella pneumoniae Vibrio vulnificus

Klebsiella oxytoca Xanthomonas campestris

Klebsiella terrigena Yersinia enterocolitica (2) Organism Serovars Organism Serovars

Kocuria kristinae Yersinia frederiksenii

Kurthia zopfii Yersinia kritensenii

Example 6: Amplification of ssrA Consensus Sequence #1 and Hybridisation of Molecular Beacon Probe #1 in Real Time

PCR amplification was undertaken using the PCR Mix shown in Table 8 (below) and the PCR program shown in Table 5 (above). The intensity of fluorescence emitted by the fluorophore component of the molecular beacon was detected at the annealing stage of each amplification cycle. In Table 8, note that the PCR buffer contains 2.25 rnM magnesium chloride (final concentration). Inclusion of additional magnesium chloride brings the final concentration to 4 mM in the reaction mixture.

Table 8. PCR Mix

Fluorescence was detected in real-time using a fluorescence monitoring real-time PCR instrument, for example, a BioRad iCycler iQ™ or MJ Research Opticon™.

Example 7: Postive Validation of Forward Primer #1, Reverse Primer #1 and Molecular Beacon Probe #1 for Detection of Listeria The effectiveness of molecular beacon probe #1, forward primer #1 and reverse primer #1 for amplification and detection of Listeria isolates was demonstrated as described generally below.

Genomic DNA from the species and strains of Listeria presented in Table 6 (see above) was isolated as described in Example 4, and amplified and detected using forward primer #1, reverse primer #1 and molecular beacon probe #1 as described in Example 6. The molecular beacon probe #1 was capable of detecting all 62 Listeria isolates tested.

Example 8; Negative Validation of the Primers and Molecular Beacon Probe #1

In order to test the ability of molecular beacon probe #1 to preferentially detect only Listeria, a number of bacteria from groups other than Listeria were tested, as generally described below.

Samples of genomic DNA from the bacteria presented in Table 9 below were isolated as described in Example 4, and amplified and detected using forward primer #1, reverse primer #1 and molecular beacon probe #1 as described in Example 6. No hybridization of this molecular beacon was observed.

In Table 9, the figures in parentheses indicate the number of strains of each species that were tested (if more than one). None of the tested strains provided a positive result.

The above results suggest that forward primer #1, reverse primer #1, and molecular beacon #1 are highly specific for Listeria species.

Table 9. Negative Validation of Molecular Beacon Probe #1, Forward Primer #1 and Reverse Primer #1

Example 9: Determination of a Second Consensus Sequence in Listeria Species ssrA Gene Sequences

An alignment of the coding region of the ssrA gene from 14 different Listeria isolates was conducted as described in Example 1. Figure 4 depicts a sample of such an alignment. A 119 nucleotide conserved sequence (SEQ ID NO:42) was identified from this alignment.

CACTYTAAGTGGGCTACACTNRNTAATCTCCGTCTGRGGTTARNTAGAAG AGCTTAATCAGACTAGCTGAATGGAAGCCTGTTACCGGGCNGANGTTTAT GCGAAATRCTAATACGGTG [SEQ ID NO:42]

This unique and conserved element of ssrA Listeria gene sequences was designated ssrA consensus sequence #2.

Example 10; Generation of DNA Primers for Amplification of ssrA Consensus Sequence #2 Within the conserved 119 nucleotide sequence identified as described in Example 8, regions that could serve as primer target sequences were identified. These primer target sequences were used to design a pair of primers to allow efficient PCR amplification of consensus sequence #2. The primer sequences are shown below:

Forward primer #3: 5'-CACTY^*TAAGTGGGCTACACT-3' [SEQ ID NO:44]

Reverse primer #3: 5'-CACCGTATTAGY⁴ATTTCGCATAAAC-S' [SEQ ID NO:45] * indicates C or T

In the alignment presented in Figure 4, the positions of forward primer #3 and reverse primer #3 are represented by shaded boxes. Forward primer #3 starts at position 145 and ends at position 164 of the alignment. Reverse primer #3 represents the reverse complement of the region starting at position 239 and ending at position 273.

Example 11; Generation of a Molecular Beacon Probe Specific for ssrA Consensus Sequence #2

In order to design molecular beacon probes specific for Listeria, a region within consensus sequence #2 was identified which not only was highly conserved in all Listeria isolates but was also exclusive to Listeria isolates. This sequence consisted of a 24 nucleotide region that would be suitable for use as a molecular beacon target sequence. The sequence is provided below:

5'- AGACTAGCTGAATGGAAGCCTGTT -3' [SEQ ID NO:43]

The complement of this sequence is also suitable for use as a molecular beacon target sequence [SEQ ID NO:49].

5'-AACAGGCTTCCATTCAGCTAGTCT-S' [SEQ ID NO:49]

A molecular beacon probe having the sequence shown below was synthesized by Integrated DNA Technologies Inc., as described in Example 3.

Molecular beacon probe #3: 5'- ccggcAGACTAGCTGAATGGAAGCCTGTTgccgg -3' [SEQ ID NO:46]

The complement of this sequence (SEQ ID NO:48, shown below) can also be used as a molecular beacon probe for the detecting Listeria.

5'- ccggcAACAGGCTTCCATTCAGCTAGTCTgccgg -3' [SEQ ID NO:48]

Table 10 provides a general overview of the characteristics of molecular beacon probe #3. The beacon sequence shown in Table 10 indicates the stem region in lower case and the loop region in upper case.

Table 10. Description of Molecular Beacon Probe #3

Beacon Sequence (5¹ -> 3'): ccggcAGACTAGCTGAATGGAAGCCTGTTgccgg

Fluorophore (5'): FAM

Quencher (3'): DABCYL

Table 11 provides an overview of the thermodynamics of the folding^'of molecular beacon probe #3. Calculations were made using MFOLD™ software, or the Oligo Analyzer software package available on Integrated DNA Technologies Inc. web site. Figure 5 shows the arrangement of the PCR primers and molecular beacon probe #3 on ssrA consensus sequence #2. Numbers in parentheses indicate the positions of the first and last nucleotides of each feature on the PCR product generated with the forward and reverse primers.

Table 11. Thermodynamics of Molecular Beacon Probe #3

Example 12: Positive Validation of Forward Primer #3, Reverse Primer #3 and Molecular Beacon Probe #3

The effectiveness of ssrA forward primer #3, reverse primer #3 and molecular beacon probe #3 for amplifying and detecting Listeria isolates was demonstrated as described generally below.

Genomic DNA from various Listeria strains was isolated as described in Example 4. Amplification was conducted as described in Example 6 with the exception that ssrA forward primer #3, reverse primer #3, molecular beacon probe #3 and the following PCR mix were used.

Table 12. PCRMix

Of the 79 strains of Listeria tested, 75 gave a positive signal (shown in Table 13). Four Listeria grayii strains gave a negative signal {i.e. sensitivity of 94.9%).

Table 13. Positive Validation of Moleculr Beacon Probe #3, Forward Primer #3 and Reverse Primer #3

Example 13: Negative Validation of Forward Primer #3, Reverse Primer #3 and Molecular Beacon Probe #3

In order to test the ability of the ssrA forward, primer #3, reverse primer #3 and molecular beacon #3 to preferentially amplify and detect only Listeria, a number of bacteria other than Listeria were tested.

Samples of genomic DNA from the bacteria presented in Table 14 below were isolated and amplified as described in the preceding Example. When no probe was included in the amplification reaction, any amplicons produced were detected using SYBR^® Green as described in Example 5. Only one amplification product was observed from the 244 strains tested {i.e. specificity of 99.6%).

Also included in additional rounds of tests was ssrA molecular beacon probe #3. Only one false positive was observed from the 244 strains tested {i.e. specificity of 99.6%).

These results suggest that both the amplification primers and the molecular beacon #3 are highly specific for Listeria.

Table 14. Negative Validation of Forward Primer #3, Reverse Primer #3 and Molcular Beacon Probe #3

Example 14; Determination of a Third Consensus Sequence in Listeria Species ssrA Gene Sequences

An alignment of the coding region of the ssrA gene from 14 different Listeria isolates was conducted as described in Example 1. Figure 7 depicts a sample of such an alignment in which regions of the non-coding strand have been aligned. A 115 nucleotide conserved sequence (SEQ ID NO:64) was identified from this alignment.

AGCTAGTCTGATTAWGCTCTTCTA(GZTZA)YTAACCYCAGACGGAGATTA(G ZTZA)Y(CZTZG)AGTGTAGCCCACTTARAGTGAGACCCTTACCGTAGCACATG GGCGATGCACGGAGGATCAGCTATKC [SEQ ID NO:64]

This unique and conserved region of the ssrA Listeria gene sequences was designated consensus sequence #3.

Example 15: Generation of DNA Primers for Amplification of ssrA Consensus Sequence #3 Within consensus sequence #3 two regions that could serve as primer target sequences were identified. These primer target sequences were used to design a pair of primers to allow efficient PCR amplification of this sequence. The primer sequences are shown below:

Forward primer #4: 5'-AGCTAGTCTGATTAW*GCTCTTCY'-3' [SEQ ID NO:66]

Reverse primer #4: 5'-ATAGCTGATCCTCCGTGC-S ' [SEQ ID NO:67]

Two further primer target sequences were identified and used to design a second pair of primers to allow efficient PCR amplification of this sequence. The primer sequences are shown below:

Forward primer #5: 5'-AGCTAGTCTGATTAW^*GCTCTTC-3' [SEQ ID NO:72]

Reverse primer #5: 5'-1VfATAGCTGATCCTCCGTG-S' [SEQ ID NO: 73]

The following reverse primer was also be used in combination with forward primer #5 to amplify consensus sequence ^3:

Reverse primer #6: 5'-GlSfATAGCTGATCCTCCGT-S' [SEQ ID NO: 74] * indicates A or T Tf indicates T or C ± indicates A or C

In the alignment presented in Figure 7, the positions of forward primer #5 and reverse primer #6 are represented by shaded boxes. Forward primer #5 starts at position 66 and ends at position 87 of the alignment. Reverse primer #6 represents the reverse complement of the region starting at position 164 and ending at position 181.

Example 16; Molecular Beacon Probes Specific for ssrA Consensus Sequence #3

hi order to design molecular beacon probes specific for Listeria, a region within consensus sequence #3 was identified which not only was highly conserved in all Listeria isolates but was also exclusive to Listeria isolates. This sequence consisted of a 23 nucleotide region that would be suitable for use as a molecular beacon target sequence. The sequence is provided below:

5 '-TGAGACCCTTACCGTAGCACAT-S ' [SEQ ID NO:65]

The complement of this sequence is also suitable for use as a molecular beacon target sequence [SEQ ID NO:71].

5'-ATGTGCTACGGTAAGGGTCTCA-S' [SEQ ID NO:71]

A molecular beacon probe having the sequence shown below and labelled with FAM at the 5' end and DABCYL at the 3' end was synthesized, as described in Example 3:

Molecular beacon probe #4:

5'-cgcgtTGAGACCCTTACCGTAGCACATaacgcg-3' [SEQ ID NO:68]

The complement of this sequence (SEQ ID NO:70, shown below) can also be used as a molecular beacon probe for the detecting Listeria.

5'-cgcgttATGTGCTACGGTAAGGGTCTCAacgcg -3' [SEQ ID NO:70]

Table 15 provides an overview of the thermodynamics of the folding of molecular beacon probe #4. Calculations were made using MFOLD™ software, or the Oligo Analyzer software package available on Integrated DNA Technologies Inc. web site. Figure 8 shows the arrangement of the PCR primers and molecular beacon probe #4 on the ssrA consensus sequence #3. Numbers in parentheses indicate the positions of the first and last nucleotides of each feature on the PCR product generated with the forward primer #4 and reverse primer #4.

Table 15. Thermodynamics of Molecular Beacon Probe #4

Example 17; Postive Validation of Forward Primer #4 and Reverse Primer #4 for Detection of Listeria

The effectiveness of forward primer #4 and reverse primer #4 for amplification and detection of Listeria isolates was demonstrated as described generally below.

Genomic DNA from the Listeria species and strains presented in Table 16 below was isolated and amplified as described in the preceding Examples (4 and 5). Results are presented in Table 16 and indicate that forward and reverse primer #4 were capable of detecting all Listeria isolates tested.

In Table 16, figures in parentheses indicate the number of strains of each Listeria serotype that were tested (if more than one). All strains gave a positive signal.

Table 16. Postive Validation of Forward Primer #4 and Reverse Primer #4

Example 18; Negative Validation of Forward Primer #4 and Reverse Primer #4

In order to test the ability of the ssrA forward primer #4, reverse primer #4 to preferentially amplify and detect only Listeria, a number of bacteria other than Listeria were tested.

Samples of genomic DNA from the bacteria presented in Table 17 below were isolated and amplified as described in the Examples 4 and 5. No amplification product was observed for the 333 strains tested {i.e. specificity of 100.0%)

These results suggest that forward primer #4 and reverse primer #4 are highly specific ϊoτ Listeria. Table 17. Negative Validation of Forward Primer #4 and Reverse Primer #4

Organism Serovars Organism Serovars

Acinetobacter calcoaceticus (2) Lactobacillus pentosus

Acinetobacter baumannii Lactobacillus plantarum

Acinetobacter iwoffi Lactobacillus rhamnosus

Acinetobacter junii Lactobacillus sakei

Aeromonas hydrophila (2) Lactobacillus salivarius

Aeromonas sahnonicida (2) Lactobacillus sanfranciscensis

Alcaligenes faecalis Lactobacillus vaginalis

Bacillus amyloliquefaciens (2) Lactococcus lactis (3)

Bacillus brevis Lactococcus raffinolactis

Bacillus cereus (2) Listeria grayi

Bacillus circulans (2) Listeria innocua (2)

Bacillus coagulans (2) Listeria ivanovii (2)

Bacillus firmus Listeria monocytogenes (2)

Bacillus lentus Listeria seeligeri

Bacillus licheniformis Listeria welshimeri

Bacillus megaterium (2) Moraxella sp.

Bacillus mycoides Micrococcus luteus

Bacillus pumilus (2) Mycobacterium smegmatis

Bacillus stearothermophilus (2) Mycobacterium fortuitum

Bacillus sphaericus Neisseria gonorrhoeae

Bacillus subtilis (2) Neisseria lactamica

Bacillus thuringiensis (2) Neisseria meningitidis

Bacteroides fragilis Neisseria sica

Bifidobacterium adolesceniis Nocardia asteroides

Bifidobacterium animalis Pantoea agglomerans

Bifidobacterium bifidum Pediococcus acidilactici

Bifidobacterium longum Pediococcus pentosaceus

Bifidobacterium sp. Proteus mirabilis (2)

Bifidobacterium suis Proteus penneri (2)

Bifidobacterium thermophilus Proteus vulgaris (2)

Branhamella catarrhalis Proteus hauseri

Brevibacillus laterosporus Providencia alcalifaciens Organism Serovars Organism Serovars

Brevundimonas diminuta Providencia rettgeri

Burkholderia cepacia (2) Pseudomonas aeruginosa (2)

Campylobacter coli (2) Pseudomonas agarici

Campylobacter jejuni (2) Pseudomonas alcaligenes

Campylobacter lari (2) Pseudomonas asplenii

Campylobacter fetus (2) Pseudomonas chlororaphis

Campylobacter hyointestinalis Pseudomonas cichorii

Campylobacter mucosalis Pseudomonas corrugata

Campylobacter rectus Pseudomonas fluorescens

Campylobacter upsaliensis Pseudomonas fragi

Campylobacter sputorum Pseudomonas marginalis

Citrobacter amalonaticus (2) Pseudomonas mendocina

Citrobacter diversus Pseudomonas oleovorans

Citrobacter freundii (S) Pseudomonas pseudoalcaligenes

Citrobacter koseri Pseudomonas fuscovaginae

Citrobacter werhnanii Pseudomonas putida (2)

Citrobacter braakii (2) Pseudomonas stutzeri

Chromobacterium violaceum Pseudomonas syringae

Chryseobacterium sp. Pseudomonas taetrolens

Chryseomonas luteola Pseudomonas tolaasii

Clostridium absonum Psychrobacter phenylpyruvicus

Clostridium bifermentans Raoultella planticola

Clostridium botulinum (2) Raoultella terrigena

Clostridium butyricum Salmonella agona

Clostridium difficile Salmonella arizonae (2)

Clostridium hastiforme Salmonella bongori

Clostridium haemolyticum Salmonella brandenburg

Clostridium novyii Salmonella choleraesuis (2)

Clostridium paraperfringens Salmonella diarizonae

Clostridium perfringens (2) Salmonella dublin (2)

Clostridium sordellii Salmonella enteritidis (2)

Clostridium sporogenes Salmonella heidelberg (2)

Clostridium spiroforme Salmonella houtenae Organism Serovars Organism Serovars

Clostridium subterminale Salmonella indica

Clostridium tetani Salmonella infantis

Clostridium tertium Salmonella montevideo

Clostridium thermosaccharolyticum Salmonella newport (2)

Clostridium tyrobutyricum Salmonella paratyphi (2)

Corynebacterium diphteriae Salmonella saintpaul

Corynebacterium xerosis Salmonella senftenberg

Edwardsiella tarda Salmonella Stanley

Enterobacter aerogenes (2) Salmonella thompson

Enterobacter amnigenus Salmonella typhimurium (2)

Enterobacter cloacae (2) Salmonella typhi (2)

Enterobacter gergoviae Salmonella typhisuis (2)

Enterobacter intermedius Serratia liquefaciens (2)

Enterobacter sakazakii Serratia marcescens (2)

Enterobacter taylorae Serratia odorifera

Enterococcus faecalis (2) Shigella boydii (2)

Enterococcus faecium Shigella dysenteriae (2)

Enterococcus hirae (2) Shigella flexneri (2)

Escherichia blattae Shigella sonnei (2)

Escherichia coli (4) Staphylococcus aureus (2)

Escherichia fergusonii Staphylococcus auricularis

Escherichia hermannii Staphylococcus chromogenes

Escherichia vulneris Staphylococcus caprae

Erwinia herbicola Staphylococcus epidermidis (2)

Geobacillus stearothermophilus Staphylococcus hyicus

Haemophilus . equigenitalis Staphylococcus intermedius

Haemophilus gallinarum Staphylococcus lentis

Haemophilus influenzae (2) Staphylococcus ludgdunensis

Haemophilus paragallinarum Staphylococcus saccharolyticus

Hafnia alvei (2) Staphylococcus saprophytics

Helicobacter cinaedi (2) Staphylococcus schieiferi

Helicobacter fennelliae Staphylococcus xylosus

Helicobacter hepaticus Stenotrophomonas maltophilia Organism Serovars Organism Serovars

Helicobacter pylori Streptococcus agalactiae (2)

Klebsiella ornithinolytica Streptococcus bovis

Klebsiella oxytoca (2) Streptococcus mitis

Klebsiella planticola Streptococcus mutans

Klebsiella pneumoniae (2) Streptococcus pneumoniae (2)

Klebsiella terήgena Streptococcus pyogenes (2)

Kocuria kristinae Streptococcus salivarius

Kocuria rosea Streptococcus sanguinis

Kocuria varians Streptococcus ihermopliilus

Kurthia zopfli Streptomyces griseus

Kurthia zopfii Vibrio alginolyticus

Legionella pneumophila (2) Vibrio campbellii

Lactobacillus acidophilus Vibrio cholerae (2)

Lactobacillus alimentarius Vibrio fischeri

Lactobacillus animalis Vibrio fluvialis

Lactobacillus aviarius Vibrio furnissii

Lactobacillus bifermentans Vibrio hollisae

Lactobacillus brevis Vibrio metschnikovii

Lactobacillus buchneri Vibrio mimicus

Lactobacillus casei Vibrio natriegens

Lactobacillus collinoides Vibrio parahaemolyticus

Lactobacillus coryniformis Vibrio parahaemolyticus

Lactobacillus curvatus Vibrio vulnificus

Lactobacillus delbreuckii Vibrio vulnificus

Lactobacillus farciminis Xanthomonas campestris

Lactobacillus fermentum Yersinia bercovieri

Lactobacillus fructivorans Yersinia enterocolitica

Lactobacillus gasseri Yersinia enterocolitica

Lactobacillus helveticus Yersinia frederiksenii

Lactobacillus hilgardii Yersinia intermedia

Lactobacillus johnsonii Yersinia kritensenii

Lactobacillus keflri Yersinia pseudotuberculosis

Lactobacillus malefermentans Yersinia pseudotuberculosis Organism Serovars Organism Serovars

Lactobacillus mali Yersinia rhodei

Lactobacillus paracasei Yersinia ruckeri

Example 19: Postive Validation of Forward Primer #5, Reverse Primer #6 and Molecular Beacon #4

The effectiveness of ssrA forward primer #5, reverse primer #6 and molecular beacon probe #4 for amplifying and detecting Listeria isolates was demonstrated as described generally below.

Genomic DNA from various Listeria strains was isolated as described in Example 4. Amplification was conducted as described in Example 6 with the exception that ssrA forward primer #5, reverse primer #6 and molecular beacon probe #4 were used.

Table 18. Positive Validation of Forward primer #5, Reverse primer #6 and Molecular Beacon Probe #4

Example 20: Negative Validation of Forward primer #5, Reverse primer #6 and Molecular Beacon Probe #4

In order to test the ability of the ssrA forward primer #5, reverse primer #6 and molecular beacon #4 to preferentially amplify and detect only Listeria, a number of bacteria other than Listeria were tested.

Samples of genomic DNA from the bacteria presented in Table 19 below were isolated and amplified as described in the preceding Example. When no probe was included in the amplification reaction, any amplicons produced were detected using SYBR^® Green as described in Example 5. No amplification products were observed from the 368 strains tested {i.e. specificity of 100%). Also included in additional rounds of tests was ssrA molecular beacon probe #3. No false positives were observed {i.e. specificity of 100%).

These results indicate that both the amplification primers and the molecular beacon #4 are highly specific for Listeria.

Table 19. Negative Validation of Forward primer #5, Reverse primer #6 and Molecular Beacon Probe #4

Organism Serovars Organism Serovars

Λcinetobacter calcoaceticus (2) Lactobacillus kefiri

Acinetohacter baumannii Lactobacillus malefermentans

Λcinetobacter iwoffi Lactobacillus mali

Acinetobacter junii Lactobacillus paracasei

Aeromonas hydrophila (2) Lactobacillus pentosus

Aeromonas salmonicida (2) Lactobacillus plantarum

Alcaligenes faecalis Lactobacillus rhamnosus

Bacillus amyloliquefaciens (2) ^; Lactobacillus sakei

Bacillus brevis Lactobacillus salivarius

Bacillus cereus (2) Lactobacillus sanfranciscensis

Bacillus circulans (2) Lactobacillus vaginalis

Bacillus coagulans (2) Lactococcus lactis (S)

Bacillus firmus Lactococcus rajfinolactis

Bacillus lentus Moraxella sp.

Bacillus licheniformis Micrococcus luteus

Bacillus megaterium (2) Mycobacterium smegmatis

Bacillus mycoides Mycobacterium fortuitum

Bacillus pumilus (2) Neisseria gonorrhoeae

Bacillus stearothermophilus (2) Neisseria lactamica

Bacillus sphaericus Neisseria meningitidis

Bacillus subtilis (2) Neisseria sica

Bacillus thuringiensis (2) Nocardia asteroides

Bacteroides fragilis Pantoea agglomerans

Bifidobacterium adolescentis Pediococcus acidilactici

Bifidobacterium animalis (2) Pediococcus pentosaceus Organism Serovars Organism Serovars

Bifidobacterium bifidum Proteus mirabilis (2)

Bifidobacterium longum (2) Proteus penneri (2)

Bifidobacterium sp. (44) Proteus vulgaris (2)

Bifidobacterium suis (2) Proteus hauseri

Bifidobacterium Tlιermophilus(2) Providencia alcalifaciens

Branhamella catarrhalis Providencia rettgeri

Brevundimonas diminuta Pseudomonas aeruginosa (2)

Burkholderia cepacia (2) Pseudomonas agarici

Campylobacter coli (2) Pseudomonas alcaligenes

Campylobacter jejuni (2) Pseudomonas asplenii

Campylobacter lari (2) Pseudomonas chlororaphis

Campylobacter fetus (2) Pseudomonas cichorii

Campylobacter hyointestinalis Pseudomonas corrugata

Campylobacter mucosalis Pseudomonas fluorescens

Campylobacter rectus Pseudomonas fragi

Campylobacter upsaliensis Pseudomonas marginalis

Campylobacter sputorum Pseudomonas mendocina

Citrobacter amalonaticus (2) Pseudomonas oleovorans

Citrobacter diversus Pseudomonas pseudoalcaligenes

Citrobacter freundii (3) Pseudomonas fuscovaginae

Citrobacter koseri Pseudomonas putida (2)

Citrobacter werkmanii Pseudomonas stutzeri

Citrobacter braakii (2) Pseudomonas syringae

Chromobacterium violaceum Pseudomonas taetrolens

Chryseobacterium sp. Pseudomonas tolaasii

Chryseomonas luteola Psychrobacter phenylpyruvicus

Clostridium absonum Raoultella planticola

Clostridium bifermentans Raoultella terrigena

Clostridium botulinum (2) Salmonella agona

Clostridium butyricum Salmonella arizonae (2)

Clostridium difficile Salmonella bongori

Clostridium hastiforme Salmonella brandenburg

Clostridium haemolyticum Salmonella ciioleraesuis (2) Organism Serovars Organism Serovars

Clostridium novyii Salmonella diarizonae

Clostridium paraperfringens Salmonella dublin (2)

Clostridium perfringens (2) Salmonella enteritidis (2)

Clostridium sordellii Salmonella heidelberg (2)

Clostridium sporogenes Salmonella houtenae

Clostridium spiroforme Salmonella indica

Clostridium subterminale Salmonella infantis

Clostridium tetani Salmonella montevideo

Clostridium tertium Salmonella newport (2)

Clostridium thermosaccharolyticum Salmonella paratyphi (2)

Clostridium tyrobutyricum Salmonella saintpaul

Corynebacterium diphteriae Salmonella senftenberg

Corynebacterium xerosis Salmonella Stanley

Edwardsiella tarda Salmonella thompson

Enterobacter aerogenes (2) Salmonella typhimurium (2)

Enterobacter amnigenus Salmonella typhi (2)

Enterobacter cloacae (2) Salmonella typhisuis (2)

Enterobacter gergoviae Serratia liquefaciens (2)

Enterobacter intermedins Serratia marcescens (2)

Enterobacter sakazakii Serratia odorifera

Enterobacter taylorae Shigella boydii (2)

Enterococcus faecalis (2) Shigella dysenteriae (2)

Enterococcus faecium Shigella flexneri (2)

Enterococcus hirae (2) Shigella sonnei (2)

Escherichia blattae Staphylococcus aureus (2)

Escherichia coli (4) Staphylococcus auricularis

Escherichia fergusonii Staphylococcus chromogenes

Escherichia hermannii Staphylococcus caprae

Escherichia vulneris Staphylococcus epidermidis (2)

Erwinia herbicola Staphylococcus hyicus

Haemophilus equigenitalis Staphylococcus intermedins

Haemophilus gallinarum Staphylococcus lentis

Haemophilus influenzae (2) Staphylococcus saccharolyticus Organism Serovars Organism Serovars

Haemophilus paragallinarum Staphylococcus saprophyticus

Hafnia alvei (2) Staphylococcus schieiferi

Helicobacter cinaedi (2) Staphylococcus xylosus

Helicobacter fennelliae Stenotrophomonas maltophilia

Helicobacter hepaticus Streptococcus agalactiae (2)

Helicobacter pylori Streptococcus bovis

Klebsiella ornithinolytica Streptococcus mitis

Klebsiella oxytoca (2) Streptococcus mutans

Klebsiella Planticola Streptococcus pneumoniae (2)

Klebsiella pneumoniae (2) Streptococcus pyogenes (2)

Klebsiella terrigena Streptococcus salivarius

Kocuria kristinae Streptococcus sanguinis

Kocuria rosea Streptococcus thermophilus

Kocuria varϊans Streptomyces griseus

Kurthia zopfii (2) Vibrio alginolyticus

Legionella pneumophila (2) Vibrio campbellii

Lactobacillus acidophilus Vibrio cholerae (2)

Lactobacillus alimentarius Vibrio flscheri

Lactobacillus animalis Vibrio fluvialis

Lactobacillus aviarius Vibrio furnissii

Lactobacillus bifermentans Vibrio hollisae

Lactobacillus brevis Vibrio metschnikovii

Lactobacillus buchneri Vibrio mimicus

Lactobacillus casei Vibrio natriegens

Lactobacillus collinoides Vibrio parahaemolyticu(2)

Lactobacillus coryniformis Vibrio vulnifιcus(2)

Lactobacillus curvatus Yersinia bercovieri

Lactobacillus delbreuckii Yersinia enterocolitica (2)

Lactobacillus farciminis Yersinia frederiksenii

Lactobacillus fermentum Yersinia intermedia

Lactobacillus fructivorans Yersinia kritensenii

Lactobacillus gasseri Yersinia pseudotuberculosis (2)

Lactobacillus helveticus Yersinia rhodei Organism Serovars Organism Serovars

Lactobacillus hilgardii Yersinia ruckeri

Lactobacillus johnsonii

Example 21: Quantification of Target Sequence in a Sample

In order to quantify the amount of target sequence in a sample, DNA wasϊsolated and amplified as described in the preceding Examples (4 and 6). DNA was quantified using a standard curve constructed from serial dilutions of a target DNA solution of known concentration.

Example 22: Testing Combinations of ssrA Specific Primers and Molecular Beacon Probes

The combination of forward primer #1, reverse primer #1, molecular beacon probe #2 (for amplification and detection of consensus sequence #1) with forward primer #3, reverse primer #3, and molecular beacon probe #3 (for amplification and detection of consensus sequence #2) were tested for sensitivity against a panel of 79 Listeria strains (as listed in Table 16) and for specificity against a panel of 244 other bacterial species.

For multiplex PCR, the protocol in Table 5 was used together with the following PCR mix (Table 20). The final concentration OfMgCl₂ in the reaction mix is 4mM.

All the 79 strains of Listeria tested gave a positive signal (i.e. sensitivity of 100%). Only two out of the 244 amplified non-Listeria strains gave a positive signal (i.e. specificity of 99.2%).

Table 20. Multiplex PCR Reaction Mix

Example 23: Specificity and Sensitivity of Primers and Molecule Beacon Probes

For testing described in this Example that uses one pair of primers alone or in combination with a single beacon, the PCR mix described in Tables 8 and 12 (above) and the PCR protocol described in Table 5 (above) were used.

When a molecular beacon was not included in the reaction, amplicons were detected with SYBR^® Green. An example of a suitable reaction mix for use with SYBR^® Green is provided in Table 4 (dNTPs and Taq polymerase are included in the Qiagen SyBrGreen Mix).

23.1 Specificity and Sensitivity of Primers

The sensitivity of the primer pair ssrA forward primer #l/reverse primer #1 was tested against a panel of 62 Listeria strains using the SYBR^® Green Reaction Mix shown in Table 4. The primer pair amplified 100% of the panel of Listeria strains.

A summary of the sensitivity and specificity of the ssrA forward primer #l/reverse primer #1 pair is shown in Table 21. Table 21. Summary for ssrA forward primer #1 and reverse primer #1

Sensitivity 100.0%

Specificity 100.0%

False positives 0.0%

False negatives 0.0%

Efficiency of primer pair 100.0%

The primer pair ssrA forward primer #3/reverse primer #3 was tested against a panel of 79 strains of Listeria. None of the strains of Listeria grayi (4) tested were detected by this primer pair. 94.9% of the Listeria panel strains was amplified.

From the panel of bacterial species other than Listeria, the ssrA forward primer #3/reverse primer #3 pair amplified sequence from one Enterobacter hirae demonstrating a specificity of 99.6%. A summary of the sensitivity and specificity of the ssrA forward primer #3/reverse primer #3 pair is shown in Table 22.

Table 22. Summary for ssrA forward primer #3 and reverse primer #3

Sensitivity 94.94%

Specificity 99.6%

False positives 0.41%

False negatives 5.06%

Efficiency of primer pair 98.5%

The primer pair ssrA forward primer #4/reverse primer #4 was tested against a panel of 79 strains of Listeria. 100.0% of the Listeria panel strains was amplified. No strains of the panel of bacterial species other than Listeria, were amplified with the ssrA forward primer #4/reverse primer #4 pair demonstrating a specificity of 100.0%. A summary of the sensitivity and specificity of the ssrA forward primer #4/reverse primer #4 pair is shown in Table 23. Table 23. Summary for ssrA forward primer #4 and reverse primer #4

Sensitivity 100.0%

Specificity 100.0%

False positives 0.0%

False negatives 0.0%

Efficiency of primer pair 100.0%

The primer pair ssrA forward primer #5/reverse primer #6 was tested against a panel of 79 strains of Listeria. 100.0% of the Listeria panel strains was amplified. No strains of the panel of bacterial species other than Listeria were amplified with the ssrA forward primer #5/reverse primer #6 pair demonstrating a specificity of 100.0%. A summary of the sensitivity and specificity of the ssrA forward primer #5/reverse primer #6 pair is shown in Table 24.

Table 24. Summary for ssrA forward primer #5 and reverse primer #6

Sensitivity 100.0%

Specificity 100.0%

False positives 0.0%

False negatives 0.0%

Efficiency of primer pair 86.0%

23.2 Primer Efficiency

Efficiency of primers was determined using 50OnM and using serial dilution of Z. monocyotogenes strain from 200 000 copies of the genome to 0.2 copies. Measurements were made in triplicate.

ssrA forward primer #1 and reverse primer #1 detect as few as 2 copies per PCR reaction. ssrA forward primer #3 and reverse primer #3 detect as few as 2 copies per PCR reaction.

ssrA forward primer #4 and reverse primer #4 detect as few as 20 copies per PCR reaction.

ssrA forward primer #5 and reverse primer #6 detect as few as 20 copies per PCR reaction.

23.3 Molecular Beacon Efficiencies

Efficiencies were tested for ssrA molecular beacon #1, #2 and #3 using DNA from L. monocytogenes strain at dilutions from 1x10° (200 000 copies of the genome) to IxIO^"6 (0.2 copy of the genome). The results were as follows:

For ssrA molecular beacon #1 : Efficiency 94.7%, detection of up to 2 copies of the genome.

For ssrA molecular beacon #2: Efficiency of 100%, detection of up to 2 copies of the genome. '

For ssrA molecular beacon #3: Efficiency 90%, detection of up to 2 copies of the genome.

For ssrA molecular beacon #4: Efficiency 95.3%, detection of up to 4.5 copies of the genome.

23.4 Specificity and Sensitivity of Molecular Beacon Probes

A summary of the sensitivity and specificity of the ssrA molecular beacon probes #1, #2 and #3 individually, and molecular beacon probes #2 and #3 together, are shown in Tables 25, 26, 27, 28 and 29. . Table 25. Summary for ssrA Molecular Beacon Probe #1

Sensitivity 100.0%

Specificity 100.0%

False positives 0.0%

False Negatives 0.0%

Efficiency of beacon 100.0%

Table 26. Summary for ssrA Molecular Beacon Probe #2

Sensitivity 100.0%

Specificity 99.18%

False positives 0.0%

False Negatives 0.82%

Efficiency of beacon 99.38%

Table 27. Summary for ssrA Molecular Beacon Probe #3

Sensitivity 94.94%

Specificity 99.59%

False positives 0.41%

False Negatives 5.06%

Efficiency of beacon 98.45% Table 28. Summary for the combination of ssrA Molecular Beacon Probes #1 and

#3

Sensitivity 100.0%

Specificity 99.18%

False positives 0.82%

False Negatives 0.0%

Efficiency of beacon 99.38%

Table 29. Summary for ssrA Molecular Beacon Probe #4

Sensitivity 100.0%

Specificity 100.0 %

False positives 0.0%

False Negatives 0.0%

Efficiency of beacon 95.3%

The ssrA molecular beacon probes #1, #2 and #4 individually detected 100.0% of the panel of Listeria strains.

The ssrA molecular beacon #3 detected 94.9% of the panel of Listeria strains. Four L. grayi strains were not detected. One non-Listeria strain were detected.

Combination of the ssrA molecular beacon probe #2 with the molecular beacon probe #3 resulted in a greater overall fluorescence and sensitivity (see Table 28).

If required an upper Ct limit can be employed in the assay. An exemplary upper limit that would be appropriate for an assay employing molecular beacon #1 in combination with molecular beacon #3 would be 39. Example 24: Enrichment Procedure

A test sample can be submitted an enrichment procedure prior to DNA extraction in order to enrich the bacterial content of the sample.

The following protocol can be followed for the enrichment of a test sample:

1. Place 25 g or 25 ml of the sample in a Stomacher filter bag.

2. Add 225 ml of Listeria Enrichment Broth (LEB) to the Stomacher filter bag.

3. Homogenize the bag contents with a Stomacher instrument.

4. Incubate the stomacher filter bag at 30°C +/- 1°C for 24 hours.

5. Ensure that the contents in the stomacher filter bag are mixed properly to obtain a homogeneous sample.

6. Transfer 200 μL of the enriched cell suspension to the Palcam tube.

7. Spread the liquid over the surface of the agar by gently shaking the tube.

8. Incubate at 35⁰C +/- 1°C for 18 hours in a slanted position while keeping the agar surface facing upward. It is imperative that the agar surface be horizontal and facing upwards so that the liquid inoculum remains spread evenly over the whole surface of the agar for the entire duration of the incubation period.

9. After incubation, add 2 mL of sterile peptone water to the tube.

10. Vortex briefly to resuspend cells.

11. Proceed with the DNA extraction protocol.

Although the invention has been described with reference to certain specific embodiments, various modifications thereof will be apparent to those skilled in the art without departing from the spirit and scope of the invention as outlined in the claims appended hereto.

The disclosure of all patents, publications, including published patent applications, and database entries referenced in this specification are specifically incorporated by reference in their entirety to the same extent as if each such individual patent, publication, and database entry were specifically and individually indicated to be incorporated by reference.

Claims

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A combination of polynucleotides for amplification and detection of one or more target nucleotide sequences from a Listeria ssrA gene, said combination selected from the group of:

(a) a combination comprising a first polynucleotide primer comprising at least 7 consecutive nucleotides of the sequence as set forth in any one of SEQ ID NOs:2-15; a second polynucleotide primer comprising at least 7 consecutive nucleotides of a sequence complementary to any one of SEQ ID NOs:2-15 and a first polynucleotide probe comprising at least 7 consecutive nucleotides of the sequence as set forth in SEQ ID NO: 16, or the complement thereof;

2. The combination of polynucleotides according to claim l(a), wherein said combination further comprises a third polynucleotide primer comprising at least 7 consecutive nucleotides of the sequence as set forth in any one of SEQ TD NOs:28-30; an fourth polynucleotide primer comprising at least 7 consecutive nucleotides of a sequence complementary to any one of SEQ TD NOs:28-30 and a second polynucleotide probe comprising at least 7 consecutive nucleotides of the sequence as set forth in SEQ ID NO:42, or the complement thereof, wherein the sequence of said second polynucleotide probe is different to the sequence of said first polynucleotide probe.

3. The combination of polynucleotides according to claim l(a), wherein said first polynucleotide primer comprises at least 7 consecutive nucleotides of the sequence as set forth in SEQ E) NO:1, said second polynucleotide primer comprises at least 7 consecutive nucleotides of a sequence complementary to SEQ E) NO: 1.

4. The combination of polynucleotides according to claim l(a), wherein said first polynucleotide primer comprises at least 7 consecutive nucleotides of the sequence as set forth in SEQ E) NO: 16, said second polynucleotide primer comprises at least 7 consecutive nucleotides of a sequence complementary to SEQ E) NO:16.

5. The combination of polynucleotides according to claim l(a), wherein said first polynucleotide primer comprises at least 7 consecutive nucleotides of the sequence as set forth in SEQ TD NO: 18 or 24; said second polynucleotide primer comprises at least 7 consecutive nucleotides of the sequence as set forth in SEQ TD NO:19 or 25, and said first polynucleotide comprises at least 7 consecutive nucleotides of the sequence as set forth in SEQ TD NOs:21 or 23.

6. The combination of polynucleotides according to claim l(a), wherein said first polynucleotide primer comprises the sequence as set forth in SEQ TD NO: 18 or 24; said second polynucleotide primer comprises the sequence as set forth in SEQ ID NO: 19 or 25, and said first polynucleotide comprises the sequence as set forth in SEQ ID NOs:21 or 23.

7. The combination of polynucleotides according to claim l(b), wherein said first polynucleotide primer comprises at least 7 consecutive nucleotides of the sequence as set forth in SEQ ID NO:1, said second polynucleotide primer comprises at least 7 consecutive nucleotides of a sequence complementary to SEQ ID NO: 1.

8. The combination of polynucleotides according to claim l(b), wherein said first polynucleotide primer comprises at least 7 consecutive nucleotides of the sequence as set forth in SEQ ID NO:42, said second polynucleotide primer comprises at least 7 consecutive nucleotides of a sequence complementary to SEQ ID NO:42.

9. The combination of polynucleotides according to claim l(b), wherein said first polynucleotide primer comprises at least 7 consecutive nucleotides of the sequence as set forth in SEQ ID NO:44; said second polynucleotide primer comprises at least 7 consecutive nucleotides of the sequence as set forth in SEQ ID NO:45, and said polynucleotide probe comprises at least 7 consecutive nucleotides of the sequence as set forth in SEQ ID NOs:47 or 49.

10. The combination of polynucleotides according to claim l(b), wherein said first polynucleotide primer comprises the sequence as set forth in SEQ ID NO:44; said second polynucleotide primer comprises the sequence as set forth in SEQ ID NO:45, and said polynucleotide probe comprises the sequence as set forth in SEQ ID NOs:47 or 49.

11. The combination of polynucleotides according to claim l(c), wherein said first polynucleotide primer comprises at least 7 consecutive nucleotides of the sequence as set forth in SEQ ID NO:1, said second polynucleotide primer comprises at least 7 consecutive nucleotides of a sequence complementary to SEQ ID NO:!.

12. The combination of polynucleotides according to claim l(c), wherein said first polynucleotide primer comprises at least 7 consecutive nucleotides of the sequence as set forth in SEQ ID NO:64, said second polynucleotide primer comprises at least 7 consecutive nucleotides of a sequence complementary to SEQ ID NO-.64.

13. The combination of polynucleotides according to claim l(c), wherein said first polynucleotide primer comprises at least 7 consecutive nucleotides of the sequence as set forth in SEQ ID NO:66 or 72; said second polynucleotide primer comprises at least 7 consecutive nucleotides of the sequence as set forth in SEQ ID NO: 67, 73 or 74, and said tpolynucleotide probe comprises at least 7 consecutive nucleotides of the sequence as set forth in SEQ ID NO:69 or 71.

14. The combination of polynucleotides according to claim l(c), wherein said first polynucleotide primer comprises a sequence as set forth in SEQ ID NO.66 or 72; said second polynucleotide primer comprises a sequence as set forth in SEQ ID NO: 67, 73 or 74, and said polynucleotide probe comprises a sequence as set forth in SEQ ID NOs: 69 or 71.

15. A method of detecting Listeria in a sample, said method comprising the steps of:

(i) contacting a sample suspected of containing, or known to contain, one or more Listeria target nucleotide sequences with the combination of polynucleotides according to any one of claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 under conditions that permit amplification and detection of said target nucleotide sequence(s), and

(ii) detecting any amplified target sequence(s), wherein detection of an amplified target sequence indicates the presence of Listeria in the sample.

16. The method according to claim 15, wherein steps (i) and (ii) are conducted concurrently.

17. The method according to claim 15 or 16 further comprising a step to enrich the microbial content of the sample prior to step (i).

18. A kit for the detection of Listeria in a sample, said kit comprising a combination of polynucleotides selected from the group of:

(b) a combination comprising a first polynucleotide primer comprising at least 7 consecutive nucleotides of the sequence as set forth in any one of SEQ ID NOs:28-41 ; a second polynucleotide primer comprising at least 7 consecutive nucleotides of a sequence complementary to any one of SEQ ID NOs:28-41, and a polynucleotide probe comprising at least 7 consecutive nucleotides of the sequence as set forth in SEQ ID NO:42, or the complement thereof;

(c) a combination comprising a first polynucleotide primer comprising at least 7 consecutive nucleotides of the sequence as set forth in any one of SEQ DD NOS:50-63; a second polynucleotide primer comprising at least 7 consecutive nucleotides of a sequence complementary to any one of SEQ ID NOs:50-63, and a polynucleotide probe comprising at least 7 consecutive nucleotides of the sequence as set forth in SEQ ID NO:64, or the complement thereof, and

(d) a combination comprising two or more of the combinations of (a), (b) and (c). .

19. The kit according to claim 18(a), wherein said combination further comprises a third polynucleotide primer comprising at least 7 consecutive nucleotides of the sequence as set forth in any one of SEQ ID NOs:28-30; an fourth polynucleotide primer comprising at least 7 consecutive nucleotides of a sequence complementary to any one of SEQ ID NOs:28-30 and a second polynucleotide probe comprising at least 7 consecutive nucleotides of the sequence as set forth in SEQ ID NO:42, or the complement thereof, wherein the sequence of said second polynucleotide probe is different to the sequence of said first polynucleotide probe.

20. The kit according to claim 18(a), wherein said first polynucleotide primer comprises at least 7 consecutive nucleotides of the sequence as set forth in SEQ ID NO:1, said second polynucleotide primer comprises at least 7 consecutive nucleotides of a sequence complementary to SEQ ID NO:1.

21. The kit according to claim 18(a), wherein said first polynucleotide primer comprises at least 7 consecutive nucleotides of the sequence as set forth in SEQ ID NO: 16, said second polynucleotide primer comprises at least 7 consecutive nucleotides of a sequence complementary to SEQ ID NO: 16.

22. The kit according to claim 18 (a), wherein said first polynucleotide primer comprises at least 7 consecutive nucleotides of the sequence as set forth in SEQ ID NO: 18 or 24; said second polynucleotide primer comprises at least 7 consecutive nucleotides of the sequence as set forth in SEQ ID NO: 19 or 25, and said first polynucleotide comprises at least 7 consecutive nucleotides of the sequence as set forth in SEQ ID NOs:21 or 23.

23. The kit according to claim 18(a), wherein said first polynucleotide primer comprises the sequence as set forth in SEQ ID NO: 18 or 24; said second polynucleotide primer comprises the sequence as set forth in SEQ ID NO: 19 or 25, and said first polynucleotide comprises the sequence as set forth in SEQ ID NOs:21 or 23.

24. The kit according to claim 18(b), wherein said first polynucleotide primer comprises at least 7 consecutive nucleotides of the sequence as set forth in SEQ ID NO:1, said second polynucleotide primer comprises at least 7 consecutive nucleotides of a sequence complementary to SEQ ID NO:1.

25. The kit according to claim 18(b), wherein said first polynucleotide primer comprises at least 7 consecutive nucleotides of the sequence as set forth in SEQ ID NO:42, said second polynucleotide primer comprises at least 7 consecutive nucleotides of a sequence complementary to SEQ ID NO:42.

26. The kit according to claim 18(b), wherein said first polynucleotide primer comprises at least 7 consecutive nucleotides of the sequence as set forth in SEQ ID NO:44; said second polynucleotide primer comprises at least 7 consecutive nucleotides of the sequence as set forth in SEQ ID NO:45, and said polynucleotide probe comprises at least 7 consecutive nucleotides of the sequence as set forth in SEQ ID NOs:47 or 49.

27. The kit according to claim 18(b), wherein said first polynucleotide primer comprises the sequence as set forth in SEQ ID NO:44; said second polynucleotide primer comprises the sequence as set forth in SEQ ID NO:45, and said polynucleotide probe comprises the sequence as set forth in SEQ ID NOs:47 or 49.

28. The kit according to claim 18(c), wherein said first polynucleotide primer comprises at least 7 consecutive nucleotides of the sequence as set forth in SEQ ID NO: 1, said second polynucleotide primer comprises at least 7 consecutive nucleotides of a sequence complementary to SEQ ID NO:1.

29. The kit according to claim 18(c), wherein said first polynucleotide primer comprises at least 7 consecutive nucleotides of the sequence as set forth in SEQ ID NO: 64, said second polynucleotide primer comprises at least 7 consecutive nucleotides of a sequence complementary to SEQ ID NO: 64.

30. The kit according to claim 18(c), wherein said first polynucleotide primer comprises at least 7 consecutive nucleotides of the sequence as set forth in SEQ ID NO:66 or 72; said second polynucleotide primer comprises at least 7 consecutive nucleotides of the sequence as set forth in SEQ ID NO: 67, 73 or 74, and said tpolynucleotide probe comprises at least 7 consecutive nucleotides of the sequence as set forth in SEQ ID NO:69 or 71.

31. The kit according to claim 18(c), wherein said first polynucleotide primer comprises a sequence as set forth in SEQ ID NO:66 or 72; said second polynucleotide primer comprises a sequence as set forth in SEQ ID NO: 67, 73 or 74, and said polynucleotide probe comprises a sequence as set forth in SEQ ID NOs:69 or 71.

32. The kit according to any one of claims 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31, wherein said polynucleotide probe is labelled with a detectable label.

33. The kit according to any one of claims 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or 32 further comprising one or more amplification reagents selected from the group of: buffers, salts, enzymes, enzyme co-factors, and nucleotides.

34. A pair of polynucleotide primers for amplification of a portion of a Listeria ssrA gene, said portion being less than 250 nucleotides in length and comprising at least 50 consecutive nucleotides of the sequence set forth in any one of SEQ ID NOs:16, 42 or 64, said pair of polynucleotide primers comprising:

(a) a first polynucleotide primer comprising at least 7 consecutive nucleotides of the sequence as set forth in SEQ ID NO:1; and

(b) a second polynucleotide primer comprising at least 7 consecutive nucleotides of a sequence complementary to SEQ ID NO:1.

35. The pair of polynucleotide primers according to claim 34, wherein said first polynucleotide primer comprises at least 7 consecutive nucleotides of the sequence as set forth in SEQ ID NO: 18 or 24 and said second polynucleotide primer comprises at least 7 consecutive nucleotides of the sequence as set forth in SEQ ID NO: 19 or 25.

36. The pair of polynucleotide primers according to claim 34, wherein said first polynucleotide primer comprises the sequence as set forth in SEQ ID NO: 18 or 24 and said second polynucleotide primer comprises the sequence as set forth in SEQ ID NO: 19 or 25.

37. The pair of polynucleotide primers according to claim 34, wherein said first polynucleotide primer comprises at least 7 consecutive nucleotides of the sequence as set forth in SEQ ID NO: 44 and said second polynucleotide primer comprises at least 7 consecutive nucleotides of the sequence as set forth in SEQ ID NO:45.

38. The pair of polynucleotide primers according to claim 34, wherein said first polynucleotide primer comprises the sequence as set forth in SEQ ID NO:44 and said second polynucleotide primer comprises the sequence as set forth in SEQ ID NO:45.

39. The pair of polynucleotide primers according to claim 34, wherein said first polynucleotide primer comprises at least 7 consecutive nucleotides of the sequence as set forth in SEQ ID NO: 66 or 72 and said second polynucleotide primer comprises at least 7 consecutive nucleotides of the sequence as set forth in SEQ ID NO:67, 73 or 74.

40. The pair of polynucleotide primers according to claim 34, wherein said first polynucleotide primer comprises the sequence as set forth in SEQ ID NO:66 or 72 and said second polynucleotide primer comprises the sequence as set forth in SEQ ID NO:67, 73 or 74.

41. An isolated Listeria specific polynucleotide consisting essentially of:

(a) the sequence as set forth in SEQ ID NO: 16, SEQ ID NO:42 or SEQ ID NO:64, or a fragment of said sequence, or

(b) a sequence that is the complement of (a).

42. A polynucleotide primer of between 7 and 100 nucleotides in length for the amplification of a portion of a Listeria ssrA gene, said polynucleotide primer comprising at least 7 consecutive nucleotides of the sequence as set forth in SEQ ID NO: 16, SEQ ID NO:42 or SEQ ID NO:64, or the complement thereof.

43. The polynucleotide primer according to claim 42, wherein said polynucleotide comprises at least 7 consecutive nucleotides of the sequence as set forth in any one of: SEQ ID NOs:18, 19, 24, 25, 44, 45, 66, 67, 72, 73 or 74.

44. The polynucleotide primer according to claim 42, wherein said polynucleotide comprises the sequence as set forth in any one of: SEQ ID NOs:18, 19, 24, 25, 44, 45, 66, 67, 72, 73 or 74.

45. A polynucleotide probe of between 7 and 100 nucleotides in length for detection of Listeria, said polynucleotide probe comprising at least 7 consecutive nucleotides of the sequence as set forth in SEQ ID NO: 16, SEQ ID NO:42 or SEQ ID NO:64, or the complement thereof.

46. The polynucleotide probe according to claim 45, wherein said polynucleotide probe comprises at least 7 consecutive nucleotides of the sequence as set forth in any one of: SEQ ID NOs:21, 23, 47, 49, 69 or 71.

47. The polynucleotide probe according to claim 45, wherein said polynucleotide probe comprises the sequence as set forth in any one of: SEQ ID NOs:21, 23, 47, 49, 69 or 71.

48. The polynucleotide probe according to claim 45, wherein said polynucleotide probe is a molecular beacon probe comprising the sequence as set forth in any one of: SEQ ID NOs:20, 22, 26, 27, 46, 48, 68 or 70.

49. The polynucleotide probe according to any one 'of claims 45, 46, 47 or 48, wherein said probe further comprises a fluorophore, a quencher, or a combination thereof.