WO2010142702A1 - A rapid real-time nucleic acid based method for the detection of salmonella species - Google Patents

Abstract

The present invention relates to a rapid and accurate method for the detection of Salmonella. The method has particular application for the detection of salmonella in foodstuffs, but also finds application in other areas such as medical and other diagnostics applications. The method involves a combined enrichment / real-time PCR method for the rapid detection of Salmonella on fresh meat based on the ssrA gene.

Description

Title

A rapid real-time nucleic acid based method for the detection of Salmonella species Field of the Invention

The present invention relates to a rapid and accurate method for the detection of Salmonella. The method has particular application for the detection of salmonella in foodstuffs, but also finds application in other areas such as medical and other diagnostics applications. Background to the Invention

Salmonella is one of the most prevalent foodborne pathogens and infects over 160,000 individuals in the EU annually with an incidence rate of 35 cases per 100,000 [H]. The annual costs of food- borne Salmonella are believed to reach up to €2.8 billion per year [7]. Reports from the World Health Organisation Surveillance Programme for Control of Foodborne Infections and Intoxications in Europe, revealed the majority of outbreaks, where causative agents were reported, were caused by Salmonella serotypes [2; 3]. Salmonellae are most often associated with any raw food of animal origin which may be subject to faecal contamination, such as raw meat, poultry, fish/seafood, eggs and dairy [12]. Salmonella testing in the slaughter environment is important as intestinal pathogens are carried into the abattoir in the bowels and on the skin of the animals. Although total viable counts (TVC) and Enterobacteriaceae testing are routinely performed on fresh meat carcasses, there was no requirement to test for Salmonella contamination prior to 2006. Good hygiene practice (GHP) and a Hazard Analysis Critical Control Point (HACCP) system must be employed to ensure minimal microbial contamination of meat carcasses during slaughter [13]. Microbiological food testing is then used to validate and verify these HACCP based procedures. The traditional culture based method for the detection of Salmonella requires the use of six different growth media, three incubation temperatures, identification by biochemical tests followed by serology [4]. Inevitably, this is a labour intensive and time-consuming exercise taking 5 days for a confirmed positive result. Alternative analytical methods, in particular, more rapid methods are permissible once they have been validated against the reference method [9; 5]. A number of real-time PCR based assays for the detection of Salmonella have been developed and published in recent years [17; 18; 25; 27]. Earlier assays lacked appropriate controls such as an IAC, which is now becoming mandatory [19; 20; 27]. Others were not validated against traditional culture methods as described in ISO 16140 [5] and/or did not meet diagnostic PCR requirements outlined in ISO 22174 [10]. Although these standards are not compulsory, the protocols could encourage the international acceptance of validation results for diagnostic realtime PCR assays [26]. Commercially available real-time PCR based kits for the detection of Salmonella include the BAX System (Oxoid), LightCycler Salmonella Detection Kit (Roche) and TaqMan® Salmonella Gold Detection and Quantitation Kit (Applied Biosystems). These kits contain an IAC, are ISO accredited, AOAC approved and have been independently evaluated [14; 15]. In this study a real- time PCR assay for the detection of Salmonella on fresh meat was developed targeting the ssrA gene. The advantage of the present invention, over these prior art kits, is that the assay of the invention is more sensitive in that it can detect less than 1.5 x 10 CFU and has a specificity of 100%. Transfer messenger RNA (tmRNA, ssrA or 10Sa RNA) has been identified in all sequenced bacterial genomes [22; 1; 28]. The ssrA gene codes for a small stable RNA molecule, tmRNA, which has many diverse functions, including tagging of abnormal proteins for degradation and modulating the activity of DNA binding proteins [21].

In this study, a combined enrichment / real-time PCR method for the rapid detection of Salmonella on fresh meat, was designed, developed and validated following requirements outlined in ISO 16140 [5], ISO 6579 [4] and ISO 22174 [10] standards. The developed method had a relative accuracy of 94.9%, sensitivity of 94.7% and specificity of 100% and was performed in 26 hours. Following the recent introduction of new EU legislation for Salmonella testing of fresh meat carcasses [9], the implementation of a rapid real-time PCR-based test such as the Salmonella real-time PCR method, would be invaluable to both food testing laboratories and fresh meat producers as it would greatly reduce turn-around time of test results. Definitions

"Synthetic oligonucleotide" refers to molecules of nucleic acid polymers of 2 or more nucleotide bases that are not derived directly from genomic DNA or live organisms. The term synthetic oligonucleotide is intended to encompass DNA, RNA, and DNA/RNA hybrid molecules that have been manufactured chemically, or synthesized enzymatically in vitro. An "oligonucleotide" is a nucleotide polymer having two or more nucleotide subunits covalently joined together. Oligonucleotides are generally about 10 to about 100 nucleotides. The sugar groups of the nucleotide subunits may be ribose, deoxyribose, or modified derivatives thereof such as OMe. The nucleotide subunits may be joined by linkages such as phosphodiester linkages, modified linkages or by non-nucleotide moieties that do not prevent hybridization of the oligonucleotide to its complementary target nucleotide sequence. Modified linkages include those in which a standard phosphodiester linkage is replaced with a different linkage, such as a phosphorothioate linkage, a methylphosphonate linkage, or a neutral peptide linkage. Nitrogenous base analogs also may be components of oligonucleotides in accordance with the invention. A "target nucleic acid" is a nucleic acid comprising a target nucleic acid sequence. A "target nucleic acid sequence," "target nucleotide sequence" or "target sequence" is a specific deoxyribonucleotide or ribonucleotide sequence that can be hybridized to a complementary oligonucleotide. An "oligonucleotide probe" is an oligonucleotide having a nucleotide sequence sufficiently complementary to its target nucleic acid sequence to be able to form a detectable hybrid probe :target duplex under high stringency hybridization conditions. An oligonucleotide probe is an isolated chemical species and may include additional nucleotides outside of the targeted region as long as such nucleotides do not prevent hybridization under high stringency hybridization conditions. Non-complementary sequences, such as promoter sequences, restriction endonuclease recognition sites, or sequences that confer a desired secondary or tertiary structure such as a catalytic active site can be used to facilitate detection using the invented probes. An oligonucleotide probe optionally may be labelled with a detectable moiety such as a radioisotope, a fluorescent moiety, a chemiluminescent, a nanoparticle moiety, an enzyme or a ligand, which can be used to detect or confirm probe hybridization to its target sequence. Oligonucleotide probes are preferred to be in the size range of from about 10 to about 100 nucleotides in length, although it is possible for probes to be as much as and above about 500 nucleotides in length, or below 10 nucleotides in length. A "hybrid" or a "duplex" is a complex formed between two single-stranded nucleic acid sequences by Watson-Crick base pairings or non-canonical base pairings between the complementary bases. "Hybridization" is the process by which two complementary strands of nucleic acid combine to form a double-stranded structure ("hybrid" or "duplex"). "Complementarity" is a property conferred by the base sequence of a single strand of DNA or RNA which may form a hybrid or double-stranded DNA:DNA, RNA:RNA or DNA:RNA through hydrogen bonding between Watson-Crick base pairs on the respective strands. Adenine (A) ordinarily complements thymine (T) or uracil (U), while guanine (G) ordinarily complements cytosine (C).

The term "stringency" is used to describe the temperature, ionic strength and solvent composition existing during hybridization and the subsequent processing steps. Those skilled in the art will recognize that "stringency" conditions may be altered by varying those parameters either individually or together. Under high stringency conditions only highly complementary nucleic acid hybrids will form; hybrids without a sufficient degree of complementarity will not form. Accordingly, the stringency of the assay conditions determines the amount of complementarity needed between two nucleic acid strands forming a hybrid. Stringency conditions are chosen to maximize the difference in stability between the hybrid formed with the target and non-target nucleic acid.

'High stringency' conditions are those equivalent to binding or hybridization at 42° C. in a solution consisting of 5xSSPE (43.8g/l NaCl, 6.9 g/1 NaH₂PO₄H₂O and 1.85 g/1 EDTA, ph adjusted to 7.4 with NaOH), 0.5% SDS, 5xDenhardt's reagent and lOOμg/ml denatured salmon sperm DNA followed by washing in a solution comprising 0. IxSSPE, 1.0%SDS at 42° C. when a probe of about 500 nucleotides in length is used.

"Medium stringency' conditions are those equivalent to binding or hybridization at 42° C. in a solution consisting of 5XSSPE (43.8 g/1 NaCl, 6.9 g/1 NaH₂PO₄H₂O and 1.85 g/1 EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS, 5xDenhardt's reagent and 100 μg/ml denatured salmon sperm DNA followed by washing in a solution comprising 1. OxSSPE, 1.0% SDS at 42° C, when a probe of about 500 nucleotides in length is used.

'Low stringency' conditions are those equivalent to binding or hybridization at 42° C. in a solution consisting of 5xSSPE (43.8 g/1 NaCl, 6.9 g/1 NaH₂PO₄H₂O and 1.85 g/1 EDTA, pH adjusted to 7.4 with NaOH), 0.1% SDS, 5xDenhardt's reagent [50xDenhardt's contains per

500ml: 5g Ficoll (Type 400, Pharamcia), 5 g BSA (Fraction V; Sigma)] and 100 μg/ml denatured salmon sperm DNA followed by washing in a solution comprising 5xSSPE, 0.1% SDS at 42° C, when a probe of about 500 nucleotides in length is used. In the context of nucleic acid in-vitro amplification based technologies, "stringency" is achieved by applying temperature conditions and ionic buffer conditions that are particular to that in-vitro amplification technology. For example, in the context of PCR and real-time PCR, "stringency" is achieved by applying specific temperatures and ionic buffer strength for hybridisation of the oligonucleotide primers and, with regards to real-time PCR hybridisation of the probe/s, to the target nucleic acid for in-vitro amplification of the target nucleic acid. One skilled in the art will understand that substantially corresponding probes of the invention can vary from the referred-to sequence and still hybridize to the same target nucleic acid sequence. This variation from the nucleic acid may be stated in terms of a percentage of identical bases within the sequence or the percentage of perfectly complementary bases between the probe and its target sequence. Probes of the present invention substantially correspond to a nucleic acid sequence if these percentages are from about 100% to about 80% or from 0 base mismatches in about 10 nucleotide target sequence to about 2 bases mismatched in an about 10 nucleotide target sequence. In preferred embodiments, the percentage is from about 100% to about 85%. In more preferred embodiments, this percentage is from about 90% to about 100%; in other preferred embodiments, this percentage is from about 95% to about 100%. By "sufficiently complementary" or "substantially complementary" is meant nucleic acids having a sufficient amount of contiguous complementary nucleotides to form, under high stringency hybridization conditions, a hybrid that is stable for detection.

By "nucleic acid hybrid" or "probe:target duplex" is meant a structure that is a double-stranded, hydrogen-bonded structure, preferably about 10 to about 100 nucleotides in length, more preferably 14 to 50 nucleotides in length, although this will depend to an extent on the overall length of the oligonucleotide probe. The structure is sufficiently stable to be detected by means such as chemiluminescent or fluorescent light detection, autoradiography, electrochemical analysis or gel electrophoresis. Such hybrids include RNA:RNA, RNA:DNA, or DNA:DNA duplex molecules.

"RNA and DNA equivalents" refer to RNA and DNA molecules having the same complementary base pair hybridization properties. RNA and DNA equivalents have different sugar groups (i.e., ribose versus deoxyribose), and may differ by the presence of uracil in RNA and thymine in DNA. The difference between RNA and DNA equivalents do not contribute to differences in substantially corresponding nucleic acid sequences because the equivalents have the same degree of complementarity to a particular sequence.

By "preferentially hybridize" is meant that under high stringency hybridization conditions oligonucleotide probes can hybridize their target nucleic acids to form stable probe:target hybrids (thereby indicating the presence of the target nucleic acids) without forming stable probe :non- target hybrids (that would indicate the presence of non-target nucleic acids from other organisms). Thus, the probe hybridizes to target nucleic acid to a sufficiently greater extent than to non-target nucleic acid to enable one skilled in the art to accurately detect the presence of (for example Candida) and distinguish these species from other organisms. Preferential hybridization can be measured using techniques known in the art and described herein. By "theranostics" is meant the use of diagnostic testing to diagnose the disease, choose the correct treatment regime and monitor the patient response to therapy. The theranostics of the invention may be based on the use of an NAD assay of this invention on samples, swabs or specimens collected from the patient. Object of the Invention It is an object of the current invention to provide sequences and/or assays to detect and identify Salmonella species. The invention is particularly suitable for detecting Salmonella on foodstuffs such as fruit, vegetables, eggs, dairy produce, fish or other seafood and fresh meats, including poultry. It is aslo an object to provide a method of detecting Salmonela in other environments such as water samples, in hospitals or food preparation or processing areas, and in diagnostic applications for the testing of faeces or bodily fluids. A further object is to provide a real-time detection and assay method which can rapidly give a result. A further object is to provide a fast and accurate assay for salmonella on fresh meat carcasses, which can be carried out while the carcase is still in the abbatoir. Such a method has the advantage that rapid detection allows a contaminated carcase to be removed from the human food chain while still in the abattoir, and avoids the need for complicated tracing procedures. It is a further object to provide a method validated to the requirements outlined in ISO 16140 [5], ISO 6579 [4] and ISO 22174 [10] standards. Summary of the Invention

The present invention provides a method for the detection and identification of Salmonella comprising :-

(a) taking a sample from an object to be tested,

(b) carrying out at least one culture enrichment step on the sample,

(c) contacting the enriched sample with at least one oligonucleotide probe derived from the ssrA gene, (d) isolating nucleic acid from the sample,

(e) hybridizing under high stringency conditions any nucleic acid that may be present in the sample with the oligonucleotide to form a probe :target duplex; and

(f) determining whether a probe :target duplex is present; the presence of the duplex positively identifying the presence of Salmonella in the test sample. The method is suitable for the detection of all salmonella serovars.

Suitably the culture enrichment step in carried out in Buffered Peptone Water or Rappaport

Vasilliadis Soya Broth. In a particularly preferred embodiment the method comprises carrying out a first culture enrichment step in Buffered Peptone Water, and carrying out a second culture enrichment step in Rappaport Vasilliadis Soya Broth. The oligonucleotide probe may be selected from :-

CAAACGACGAAACCTACGCTTTGC, and

AGCGTTAAAACGAATCAGGCTAGTCT.

Preferably the test sample is contacted with both oligonucleotide probes.

Preferably the enrichment step in Buffered Peptone Water is carried out for 16 to 20 hours and preferably 18 hours. This enrichment step may be carried out at 36 to 38 ⁰C and preferably 37⁰C.

Preferably the enrichment step in Rappaport Vasilliadis Soya Broth is carried out for 4 to 8 hours and preferably 6 hours. This enrichment step may be carried out at 41 to 43 ⁰C and preferably

42⁰C. Suitably an internal amplification control which is amplified with the same primer set as the Salmonella target, is used in step (f). The internal amplification control may be an E. coli. ssrA gene fragment.

Either DNA or RNA may be isolated from the sample. DNA or RNA may be isolated from the culture supernatant of the sample. Conventional methods of DNA isolation may be used. Where RNA is isolated it may be reverse-transcribed to produce cDNA. The RNA may be treated with DNAase to remove contaminating DNA. Such methods of DNA and RNA isolation and reverse transcription are well known to those skilled in the art and several suitable kits are commercially available. The method may also comprise a step for releasing nucleic acid from any cells of the target organism that may be present in said test sample. Such methods are also well known to the skilled person.

Suitably, the sample which is analysed may be taken by swabbing the object to be tested. The swab may be used to inoculate the enrichment broth. The present invention also provides for a diagnostic kit for detection and identification of Salmonella species, comprising probes based on the ssrA gene. The probes may be selected from :- CAAACGACGAAACCTACGCTTTGC, and

AGCGTTAAAACGAATCAGGCTAGTCT. The nucleic acid molecule may be synthetic. The kit may further comprise a primer for amplification of at least a portion of the ssrA gene. Suitably the kit comprises a forward and a reverse primer for a portion of the ssrA gene. The kit may also comprise additional primers or probes. The kit may additionally comprise one or more of buffers, DNA isolation reagents and nutrient broths.

The invention also provides a nucleic acid molecule selected from the group consisting of :- CAAACGACGAAACCTACGCTTTGC, and AGCGTTAAAACGAATCAGGCTAGTCT and sequences substantially homologous thereto, or substantially complementary to a portion thereof and having a function in diagnostics based on the ssrA gene.

The nucleic acid molecule and kits of the present invention may be used in a diagnostic assay to detect the presence of Salmonella species, to measure bacterial titres in a patient or in a method of assessing the efficacy of a treatment regime designed to reduce bacterial titre in a patient or to measure bacterial contamination in an environment. The environment may be a hospital, or it may be a food sample, an environmental sample e.g. water, an industrial sample such as an in- process sample or an end product requiring bioburden or quality assessment. A particularly preferred environment is a fresh meat carcase, but could also be vegetables, salads, peanuts, sesame seeds, baby foods, sausages or the like. Brief Description of the Drawings

Figure 1 Left: SAM 1 probe region (light grey) highlighting mismatches (dark grey) with one

Salmonella serovar (S. Agona) and selected closely related species.

Right: SAM 2 probe region (light grey) highlighting mismatches (dark grey) with one Salmonella serovar (S. Anatum) and related species.

Figure 2 Quantification curve demonstrating specificity of the Salmonella real-time PCR assay

(Channel 530 nm).

Quantification curves demonstrating IAC detection (Channel 610 nm) for all samples in the same experiment with the exception of E. aerogenes NCTC 10006. Figure 3 Quantification curves demonstrating detection (Channel 530 nm) of samples spiked with

S. Typhimurium ATCC 14028 ranging from 1-5,000 CFU per 100cm² in the Salmonella real-time

PCR assay. Quantification curve demonstrating IAC detection (Channel 610 nm) for all samples in same experiment.

Detailed Description of the Invention Materials and Methods

Bacterial strains and culture conditions

A total of 30 Salmonella serovars and 30 non-Salmonella organisms were used in this study

(Table 1) for inclusivity and exclusivity testing respectively, as required by ISO 16140 [5].

Salmonella serovars were incubated at 37 ⁰C while non-Salmonella organisms were incubated at 30 ⁰C or 37 ⁰C, as appropriate.

Traditional Culture Method - ISO 6579:2002

The reference method was performed as described in ISO 6579:2002. The following materials were used: Rappaport Vasilliadis Soya (RVS; Oxoid, Basingstoke, Hampshire, UK), Muller

Kauffmann Tetrathionate with Novobiocin broth (MKTTn; AES Laboratoire, Essex, UK), Xylose-Lysine-Desoxycholate (XLD; Oxoid), Brilliant Green Agar (BGA; Oxoid), Tryptone

Soya Agar (TSA; Oxoid), API 2OE strips (Biomerieux, Hampshire, UK), API James

(Biomerieux), API TDA (Biomerieux), API VP1/VP2 (Biomerieux), Identification Sticks

Oxidase (Oxoid), Hydrogen Peroxide 4% (Sigma-Aldrich Ireland Ltd., Dublin, Ireland). Positive and negative controls (Salmonella Typhimurium ATCC 14028 and E. coli ATCC 25922 respectively) were also included.

Modified Culture Method

The modified culture enrichment method involved primary enrichment in BPW (100 ml) for 18 h at 37 ⁰C. After incubation, 100 μl of pre-enrichment culture was transferred to 10 ml RVS and incubated at 42 ⁰C for 6 h. One millilitre of Salmonella/RVS culture was then centrifuged (5,000 x g for 10 min) and the cell pellet washed in 1 ml phosphate buffered saline (PBS; Oxoid). DNA isolations were performed as described below and stored at -20 ⁰C until use. For RNA isolation, 8 ml of Salmonella/ Rappaport-Vassiliadis Soya peptone (RVS; Oxoid) culture was centrifuged (4,200 x g for 10 min) and the cell pellet washed in 8 ml PBS. RNA isolations were performed as described below and stored at -80 ⁰C until use. DNA isolation and quantification

Genomic DNA was prepared from 1 ml of overnight cultures grown in tryptic soya broth (TSB; Oxoid) or RVS. DNA isolations were performed using the DNeasy Blood and Tissue kit (Qiagen, West Sussex, UK) following manufacturer's instructions. Total genomic DNA was quantified using the TBS-380 mini-fluorometer (Turner BioSystems, Sunnyvale, California, USA) and PicoGreen dsDNA Quantitation Kit (Invitrogen Corporation, Carlsbad, California, USA). Generation of sequence data

Salmonella sequencing primers, Entero-tm F and Entero-tm R (Table 2), were designed based on the 5' and 3' flanking regions of Enterobacteriaceae tmRNA sequence [I]. These primers were used to amplify a 363 bp ssrA gene fragment from 30 Salmonella serovars and closely related organisms. Conventional PCR was performed as described below. Products were purified using the High Pure PCR Product Purification Kit (Roche Diagnostics), and sent for sequencing (Sequiserve, Vaterstetten, Germany). Sequence alignments were performed using Clustal W multiple sequence alignment programme (http://www.ebi.ac.uk /tools/clustalw/index.html). Conventional PCR

Conventional PCR was performed using the iCycler iQ thermal cycler (Bio-Rad Laboratories Inc., Hercules, California, USA). Reactions were performed in 50 μl volumes consisting of: 1OX buffer (containing 15 mM MgCl₂), 1 μl Taq DNA polymerase (IU/ μl; Roche Diagnostics Ltd., West Sussex, UK), 1 μl dNTP mix (10 mM; Deoxynucleoside Triphosphate Set - Roche Diagnostics), 1 μl forward and reverse primers (20 μM; Table 2), 39 μl nuclease free H₂O

(Applied Btosystems/Amhion, Austin, Texas, USA) and 2 μl PCR template (genomic DNA). The cycling parameters consisted of 30 cycles of denaturation at 94 ⁰C (30 sec), annealing at 50 ⁰C (60 sec) and extension at 72 ⁰C followed by a final extension at 72 ⁰C for 10 min. Primer and probe design for Salmonella real-time PCR assay Salmonella assay primers (FPF and FPR), Salmonella specific TaqMan probes (SAM 1 and SAM 2) and an IAC TaqMan probe (IAC-Entero) were designed following alignment of ssrA gene sequences of Salmonella serovars and related organisms (Table 2, Fig. 1) according to general guidelines and recommendations [16; 35]. The BLAST-N program (National Centre for Biotechnology Information; http://www.ncbi.nlm.nih.gov) was used to search nucleotide databases using query primer and probe sequences. The primers and probes were suspended in nuclease free water to a concentration of 100 μM and working stocks of 5 μM and 2 μM respectively, were prepared. All stocks were stored at -20 ⁰C. Salmonella real-time PCR assay Real-time PCR reactions were performed on the LightCycler® 2.0 Instrument (Roche

Diagnostics) using the LightCycler® FastStart DNA Master HybProbe kit (Roche Diagnostics). PCR was performed in a final volume of 20 μl including 2 μl DNA template in LightCycler hybridisation buffer with MgCl₂ adjusted to 5 mM concentration. LightCycler® Uracil-DNA Glycosylase (Roche Diagnostics) (0.3 μl) was also added to each reaction to minimise risk of contamination. PCR primers, FPF and FPR (0.5 μM final concentration) and TaqMan probes,

SAM 1, SAM 2 and IAC -Entero (0.2 μM final concentration) were added to the reaction mixture. Two micro litres of IAC plasmid (100 copies) was added and the volume was adjusted to 20 μl with nuclease free H₂O. Optimum PCR cycling conditions included incubation for 10 min at 95°C for initial denaturation and activation of the chemically modified Taq DNA polymerase (which is inactive at room temperature), followed by 50 amplification cycles with denaturation for 10 sec at 95⁰C and an annealing for 30 sec at 64°C. With a maximum emission of 520 nm, the Salmonella FAM-labelled probes were detected in Channel 1 (530 nm) while the IAC ROX-labelled probe, with a maximum emission of 602 nm was detected in Channel 3 (610 nm). Prior to sample analysis, a colour compensation file was generated on the LightCycler following the protocol outlined in Technical Note No. LC 21/2007 [31]. This file was imported into each data file generated, to compensate for "bleed-over" of fluorescence signal between channels. Real-time PCR products were run on 1.5% TBE agarose gels at 120 V for 1 h, and visualised under UV using the FluoroChem™ 8900 and AlphaEase FC software. Internal Amplification Control E. coli ssrA gene fragment was amplified using primers FPF and FPR (Table 2). The PCR product was ligated into a plasmid and cloned into chemically competent E. coli cells using the pCR®2.1- TOPO® TA cloning kit (Invitrogen) according to manufacturer's instructions. Plasmid purification was carried out using the QIAprep Spin Miniprep Kit (Qiagen) according to manufacturer's instructions. A titration was performed to determine optimum copy number to be used in the assay, such that the IAC would always be detected without affecting detection of the primary Salmonella target. Validation

The combined enrichment / real-time PCR method was validated against the traditional culture method ISO 6579 [4], in accordance with ISO 16140 [5]. Validation in pure culture

Initial validation was performed using pure culture of 30 Salmonella serovars and 30 non- Salmonella organisms. One hundred millilitres of BPW was inoculated with 1 CFU for Salmonella serovars (final concentration of 0.01 CFU/ml) and 1,000 CFU for non-Salmonella organisms (final concentration of 10 CFU/ml). Reference and alternative methods were performed in parallel as described above. Validation in spiked carcass swabs

Fresh carcass swabs were collected in local abattoirs. Four sites on the carcass were swabbed (4 x 100 cm²) using pre-moistened sterile sponge swabs (Technical Service Consultants Limited, Heywood, Lancashire, UK) i.e. neck, brisket, flank and rump for beef, and jowl, back, belly and ham for pork. Two swabs, front and back, were used to sample each carcass and then placed together in a sterile bag. A minimum of 25 carcasses was swabbed on each visit to the abattoir (i.e. one replicate). Six replicates of fresh carcass swabs (3 replicates of beef and 3 replicates of pork) were collected for this study (75 beef and 75 pork samples in total). Samples were immediately placed into a cool box on ice. Sample analysis was performed a maximum of 24 hours after sample collection.

Each carcass swab was tested for naturally occurring Salmonella according to ISO 6579 [4] and Enterobacteriaceae according to ISO 21528-2 [8] to determine the level of background contamination. Violet red bile green agar (VRBGA; Oxoid) and glucose agar (Mast Group Ltd, Merseyside, UK) were used in Enterobacteriaceae testing. Swabs containing naturally occurring Salmonella as determined by the reference method were discarded. Swabs were inoculated with 5 different Salmonella serovars (Derby, Dublin, Livingstone, Typhimurium, Typhimurium DT 104) at five inoculation levels (1, 10, 100, 1000, 5000 CFU/100 cm²), 25 samples in total. These spiked samples were then tested for the presence of Salmonella using the ISO culture based method and the alternative molecular method as previously described (Fig.3). Blind sample study

Twenty- four blind samples, in the form of spiked cotton swabs, were prepared in an independent microbiology laboratory (Ashtown Food Research Centre, Teagasc, Dublin, Ireland). Samples contained varying levels of Salmonella, with and without background Enterobacteriaceae. Five Salmonella serovars were used in the experiment (Derby SARB 11, Dublin NCTC 09676,

Livingstone NCTC 09125, Typhimurium DT 104 NCTC 13348 and Typhimurium ATCC 14028), while background flora contained E. coli NCTC 09001, Enterobacter cloacae NCTC 10005 and Citrobacter freundi NCTC 09750. Blank samples were also included in the study. All cultures were grown in nutrient broth for 24 h at 37 ⁰C. Serial 10-fold dilutions of each culture were performed and the 10^"5 (-1,000 CFU/ml), 10^"6 (-100 CFU/ml), 10^"7 (-10 CFU/ml) and 10^"8 (~1 CFU/ml) dilutions were used to inoculate the swabs. Each sterile cotton swab (Nuova Aptaca, Regione Monforte, 314053 Canelli, Italy) was inoculated with different strains at different inoculation levels (Table 3). One millilitre of each dilution was mixed together in a sterile tube. The dry cotton swab was placed in the bacterial mix and allowed to stand for 30 min. A solution of semi-solid nutrient agar was prepared using half the indicated weight per volume of powder. A sufficient volume of this semi-solid agar was dispensed into the plastic tube to ensure the cotton swab was fully immersed. These samples were shipped on ice to our laboratory and analysed using the developed Salmonella real-time PCR based method as described above. RNA isolation and quantification

RNA was prepared from 8 ml (unless stated otherwise) of bacterial cultures in TSB or RVS using the Ambion 'RiboPure- Yeast Kit' (Ambion, Texas, USA) following manufacturer's instructions and eluted in 50 μl. Total RNA was quantified using the TBS-380 mini-fluorometer (Turner BioSystems, California, USA) and RiboGreen RNA Quantitation Kit (Invitrogen Corporation, California, USA). DNase treatment of RNA

Total RNA samples were DNase treated using the 'TURBO DNA-free Kit' (Ambiors) before use in real-time RT-PCR. Whole samples (i.e. 50 μl) were treated twice to ensure complete digestion of contaminating DNA following the rigorous DNase treatment protocol supplied. cDNA synthesis and real-time PCR

The reverse transcription step of the 2-step RT-PCR procedure was performed using the RETROscript® Kit (Ambion) using a gene specific reverse primer i.e. the real-time PCR reverse primer [FPR; (McGuinness et ah, 2009)] as per manufacturers' instructions. The sample was either stored at -20 ⁰C or used immediately as template for real-time PCR. Real-time PCR was performed as described by McGuinness et al. (2009). ISO 16140 - Phase 1 Validation (pure culture)

Initial validation was performed on the modified enrichment / real-time RT-PCR method. This alternative method was validated in pure culture (Phase 1 validation) against the traditional culture method ISO 6579 (Anonymous, 2002), in accordance with ISO 16140 (Anonymous,

2003). Phase 1 validation was performed using pure cultures of 30 Salmonella strains and 30 non- Salmonella strains which were either closely related or commonly found on fresh meat (Table 4). One hundred millilitres of BPW was inoculated with ~1 CFU for Salmonella serovars (final concentration of 0.01 CFU/ml) and -1,000 CFU for non-Salmonella species/strains (final concentration of 10 CFU/ml). Following 18 h enrichment in buffered peptone water (BPW), reference and alternative methods were performed in parallel. Results

Assay design and development In-silico analysis of Salmonella ssrA sequence data revealed that no single probe would enable detection of all Salmonella serovars while maintaining specificity. One probe was designed that could detect 29/30 Salmonella serovars and a second probe was designed to detect S. Anatum. There is only one base difference between Salmonella serovars and the most closely related organisms e.g. Citrobacter, in both probe regions (Fig. 1). Salmonella probes (SAM 1 and SAM 2) were designed to have similar melting temperatures (Table 2) and the probes are labelled with the same fluorescent molecules and read in the same channel of the LightCycler. Detection of Salmonella can result from three probe combinations i.e. SAM 1 only (n = \; S. Anatum); SAM 2 only (n = 14); SAM 1 + SAM 2 (n = 15). An IAC probe was designed to detect the ssrA gene of Escherichia coli and closely related species belonging to the Enterobacteriaceae family including Salmonella. This probe region was common to all but one of the Enterobacteriaceae strains (i.e. Enterobacter aerogenes; Fig. 2) examined. One hundred copies of the IAC plasmid containing the 286 bp E. coli ssrA gene fragment was determined to be the optimum concentration for use in the real-time PCR assay. Inclusivity and exclusivity of Salmonella real-time PCR assay Inclusivity of the assay was confirmed with 100 ng genomic DNA from 30 Salmonella serovars. Included in the Salmonella real-time PCR assay, were a negative control (E.coli ATCC 25922) and a no-template control. Exclusivity of the assay was confirmed using 100 ng genomic DNA from 30 non-Salmonella organisms (Fig. 2). Included in the assay were a positive control (Salmonella Dublin NCTC 09676) and a no-template control. Inclusivity and exclusivity was not affected by the inclusion of the IAC.

Performance of the ssrA Salmonella real-time PCR assay

The Salmonella genome size is approximately 4.8 Mb [30] which is equal to ~ 5 fg DNA per cell. Based on this information, sensitivity experiments were performed using serial dilutions of Salmonella genomic DNA from 10⁶ to 10° genome equivalents. Analysis was performed in triplicate for 3 Salmonella serovars, each with a different probe binding combination i.e. S.

Dublin, S. Anatum and S. Agona. A detection limit of 1-10 genome equivalents per reaction was determined for each target species. Average PCR efficiency from 9 sensitivity experiments was 2.044 with an error of 0.148. Validation Validation in pure culture

All Salmonella serovars were detected in pure culture using a combination of shortened culture enrichment and real-time PCR. The non-Salmonella organisms were not detected using the rapid method. Sample analysis using the traditional culture method, ISO 6579, yielded identical results. Validation in spiked carcass swabs

In the presence of natural background flora, the real-time PCR method had a relative accuracy of 94.9%, sensitivity of 94.7% and specificity of 100% when compared to the traditional ISO method. Detection probabilities were 73% when carcass swabs were spiked with Salmonella at 1 CFU/100 cm², and 100% when spiked with 10 CFU/100 cm² and above (Fig. 3). Ninety five percent confidence intervals of 91.1% - 98.3% were observed. Blind sample study

With the exception of Sample 22, positive and negative samples were identified correctly using the Salmonella real-time PCR method (Table 3), i.e. results reflected inoculation data which was received post-analysis. Sample 22 contained ~1 CFU/ml of Salmonella prior to absorbance onto the swab however was undetectable in a background of ~10 CFU/ml of E. coli when analysed using the culture combined / real-time PCR method. Real-Time RT-PCR Assay

Inclusivity and exclusivity were determined using 100 ng of tmRNA cDNA from 30 Salmonella and 30 non-Salmonella strains respectively (Table 4). The limit of detection of the real-time RT- PCR assay was < 1 cell equivalent.

Validation of the alternative method was performed in pure culture according to the ISO 16140 standard. All Salmonella serovars were detected using the alternative method while the non- Salmonella strains were un-detected. All samples analysed in parallel using the traditional culture method, ISO 6579:2002, yielded identical results. Significantly earlier Ct values were observed for the real-time RT-PCR based method when compared to the real-time PCR based method [Table 5)]. On average Ct values were 9.672 cycles earlier which equates to -1,000-fold increase in assay sensitivity.

Comparison of Real-Time PCR and Real-Time RT-PCR Assays In order to make a direct comparison between sensitivity of Salmonella real-time PCR and real- time RT-PCR assays, DNA and RNA isolations were performed from equal volumes (1 ml) of a Salmonella ATCC 14028/RVS culture following culture enrichment [18 h in BPW + 6 h in RVS. DNA and RNA isolations were eluted in equal volumes (50 μl). One RNA sample was subjected to DNase treatment prior to cDNA synthesis while another RNA sample was left un-treated. cDNA was prepared from the DNase treated and un-treated RNA samples. DNA, cDNA and appropriate controls were then analysed using the ssrA Salmonella real-time PCR assay. Significantly earlier Ct values were observed when cDNA samples were compared to the DNA sample in the Salmonella real-time PCR assay (Table 6). Furthermore, cDNA prepared from total RNA un-treated with DNase was detected ~ 3 Ct's earlier than cDNA prepared from DNase treated RNA. This may be explained by the fact that DNA, co-purified with the RNA would also be detected and lead to increased sensitivity of the assay. Evidence that DNA is co-purified can be seen in Table 6 where the total RNA sample un-treated with DNase was detected by PCR alone, while the DNase treated RNA sample yielded no signal. DNase treatment may also have resulted in some RNase activity leading to a reduction in RNA concentration and subsequently later Ct values. In addition, the DNase treatment may also have inhibited the rate of reverse transcriptase cDNA conversion of tmRNA or the PCR reaction itself.

The template that produced the earliest Ct value was the cDNA prepared from total RNA untreated with DNase. In our opinion the increased sensitivity observed maybe as a consequence of the combined cDNA and ssrA gene amplification.

Also, the added time and cost of DNase treatment are significant and could therefore be removed from this method in the future.

Additional exclusivity testing of Salmonella real-time PCR assay

A total of 53 additional non-Salmonella organisms were used for exclusivity testing of the real- time PCR assay (Table 7). Exclusivity of the assay was confirmed by including 10⁴ cell equivalents of DNA extracted from the organisms in the real-time PCR assays. No positive signals were produced when tested with the 53 non-Salmonella organisms. The DNA extracted from non-Salmonella bacterial and fungal organisms was confirmed to be amenable to real-time- PCR by visualisation of PCR products and amplification with universal tm RNA and 16S rRNA (as control) primers and universal fungal primers, respectively. This additional exclusivity testing was performed using the Probes Master kit (Roche) on the LightCycler® 480 (Roche) real-time PCR instrument. Discussion The aim of this research was to develop a rapid test for the detection of Salmonella on fresh meat carcasses. Recently the ssrA gene was used as a diagnostic target for the detection of Listeria monocytogenes in enriched food samples [29]. In this study detection of the Salmonella ssrA gene using real-time PCR combined with a 24 h enrichment step, was compared to the traditional culture based method (ISO 6579:2002) [4]. There is limited heterogeneity in the ssrA gene between genera of the Enterobacteriaceae family, making specific assay design for Salmonella quite challenging. Two Salmonella specific TaqMan probes were required to achieve 100% inclusivity. As there is only one base difference between Salmonella serovars and related species in the two probe regions, the mismatch was placed in the centre of the sequence to increase probe differentiation ability and achieve 100% inclusivity (Fig.l). It has been reported that the melting temperature of a probe decrease when mismatches are present and one study has demonstrated that a C-A mismatch (the most destabilising mismatch) in the centre of a probe lowered the melting temperature by 8°C [23]. The SAM 1 probe contains a central C-A mismatch and the SAM 2 probe contains a central G-T mismatch. Negative amplification test results do not necessarily indicate absence of the target organism in the test sample [33]. A positive signal from a secondary target (i.e. an IAC) indicates successful amplification and therefore validates a negative result for the primary target [33]. An internal amplification control in each PCR reaction monitors the presence of inhibitory substances, malfunctions with the thermocycler, poor enzyme activity, incorrect reaction mixture, and false negative results [34]. An IAC can be used competitively or non-competitively in a diagnostic

PCR assay [20]. A competitive IAC is based on the composite primer technique [36] where both the target DNA and the IAC are amplified using the same primer pair. In this situation, there is always a degree of competition and therefore the optimum copy number of IAC must be determined. The presence of background flora can cause problems when detecting Salmonella in environmental samples [24]. The IAC probe was designed to detect the ssrA gene of E. coli and closely related species belonging to the Enterobacteriaceae family. As the primer set used in the Salmonella real-time PCR assay are not Salmonella specific, the presence of background Enterobacteriaceae on meat carcasses can cause downstream competition for primers between the target sequence, background Enterobacteriaceae, and the IAC. In cases where Salmonella are absent on the carcass swabs, competition arises for primers between Enterobacteriaceae and low copy numbers of the IAC. The IAC probe will bind to the ssrA sequence of the Enterobacteriaceae, thus eliminating the risk of an invalid result. The non-Salmonella panel comprised of 30 organisms, 15 of which belonged to the Enterobacteriaceae family. The IAC probe did not bind to the ssrA sequence of E. aerogenes (Fig. 2), the only organism examined from the Enterobacteriaceae family to yield an unsuccessful result in this regard. This situation was unavoidable due to sequence constraints. Due to primer competition, the IAC is not detected in the presence of a high concentration of exclusively E. aerogenes DNA, therefore yielding an invalid result for a sample that does not contain Salmonella. This situation is almost certain never to arise however, as it would be extremely unlikely to collect an environmental sample containing a pure culture of E. aerogenes. To increase the concentration of the target organism and to ensure detection of viable bacteria, samples were homogenised and enriched in non-selective BPW (1:10 dilution) followed by semi- selective enrichment in RVS (1:100 dilution). This step also dilutes possible biological contaminants such as blood and fats present on animal carcasses as well as enrichment broth or its components which can cause PCR inhibition [32]. The bacterial cell pellet was washed in PBS prior to DNA isolation to reduce the concentration of salts (Magnesium chloride (anhydrous) 13.58 g/L) and the residual blue colouring (Malachite green 0.036 g/L) from the RVS broth. Although there has been a fall in the number of human Salmonella infections in Europe over the past number of years [11], food-borne infection from Salmonella continues to pose a great risk to public health. It is widely believed that pathogen reduction in animals is the most effective way to prevent the spread of infection via food, therefore legislation has been designed to improve protective measures against zoonoses [7; 6]. Also important, are efficient Quality Control Systems (e.g. HACCP principles and good hygiene practices), and Quality Assurance (QA) at all stages of the food chain from "farm to fork". Routine QA testing of food samples is of utmost importance to monitor hygiene standards. The presence of Salmonella on fresh meat carcasses was addressed by the European Union in 2001 and new regulatory microbiological criteria was published in 2005 [9]. There is a zero tolerance in the European Union for the presence of Salmonella in all food types, from infant formula to minced meat. However, there is some deviation from such stringent regulations pertaining to animal carcasses i.e. in 10 consecutive sampling sessions (5 swabbed carcasses per session), a maximum of 2 carcass for beef or 5 carcasses for pork (from a total of 50 samples) can be reported as positive for Salmonella and yet satisfactorily comply with regulations. The real-time PCR method described in this report is designed for the rapid detection of Salmonella on fresh meat carcasses. The validation of this qualitative test complies with requirements as set down by the International Organisation for Standardisation (ISO16140: 2003). Analytical controls employed for validation in spiked carcass swabs included an amplification positive control, a no template control (NTC), and an internal amplification control (IAC). Un- inoculated BPW and RVS control broths were also processed throughout the protocol. Detection probabilities were 73% when fresh meat carcass swabs were spiked with Salmonella at 1 CFU/100 cm², and 100% when spiked with 10 CFU/100 cm² and above (Fig. 3) which compares favourably with the traditional culture method (ISO 6579). This rapid Salmonella test with a detection limit of 1-10 CFU/100 cm² in the presence of naturally occurring background flora can be performed in 26 hours. Following an inter-laboratory trial, this assay has the potential to become a standardised method for routine analysis of carcass swabs for the presence of Salmonella. The ability to detect all Salmonella serovars with no risk of false positive results while vastly reducing the analysis time would make this assay an invaluable asset both to food testing industry and food producers. This assay further demonstrates the potential of the tmRNA / ssrA gene as a platform nucleic acid based diagnostic target.

The increase in sensitivity observed between the real-time RT-PCR and real-time PCR methods indicates that targeting tmRNA may enable the shortening of the culture enrichment section of the method and/or increase the overall limit of detection of this alternative method. There is an urgent need for a rapid and specific Salmonella test with 100 % relative sensitivity to the traditional culture method, which this assay has the potential to meet.

The words "comprises/comprising" and the words "having/including" when used herein with reference to the present invention are used to specify the presence of stated features, integers, steps or components but does not preclude the presence or addition of one or more other features, integers, steps, components or groups thereof.

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. References

1. Anonymous The tmRNA Website, http://www.indiana.edu/-tmrna/.

2. Anonymous (1998) WHO Surveillance Programme for Control of Foodborne Infections and Intoxications in Europe: 7th Report 1993-1999. Edited by C. Tirado, K. Schmidt. W H O.

3. Anonymous (2000) WHO Surveillance Programme for Control of Foodborne Infections and Intoxications in Europe: 8th report 1999-2000. Edited by K. Schmidt, A. Gervelmeyer. WHO.

4. Anonymous (2002) Microbiology of food and animal feeding stuffs - Horizontal method for the detection of Salmonella spp. In EN ISO 6579:2002, pp. 1-28. Geneva, Switzerland.

5. Anonymous (2003) Microbiology of food and animal feeding stuffs - Protocol for the validation of alternative methods. In EN ISO 16140:2003, pp. 1-78. Geneva, Switzerland. 6. Anonymous (2003) Regulation (EC) 2160/2003 of the European Parliament and Council of 17 November 2003 on the control of Salmonella and other specified food-borne zoonotic agents. 7. Anonymous (2003) Zoonoses: Commissioner David Byrne welcomes new legislation to combat food-borne diseases such as Salmonella (IP/03/1306). European Commission. 8. Anonymous (2004) Microbiology of food and animal feeding stuffs. Horizontal methods for the detection and enumeration of Enterobacteriaceae. Colony-count method, Part 2. In EN ISO 21528-2:2004, Geneva, Switzerland.

9. Anonymous (2005) Commission regulation (EC) No 2073/2005 on microbiological criteria for foodstuffs. Official Journal of the European Union L338, 1-26.

10. Anonymous (2005) Microbiology of food and animal feeding stuffs - Polymerase chain reaction (PCR) for the detection of food-borne pathogens - General requirements and definitions. In EN ISO 22174:2005, pp. 1-11. Geneva, Switzerland.

11. Anonymous (2006) The Community Summary Report on Trends and Sources of Zoonoses, Zoonotic Agents, Antimicrobial Resistance and Foodborne Outbreaks in the European Union in

2006. The EFSA Journal 130, 1-310.

12. Anonymous (2007) Salmonella spp., pp. 1-5. Food Safety Authority of Ireland (FSAI).

13. Bolton, D.J., Pearse, R.A., Sheridan, J.J., Blair, I.S., McDowell, D.A. and Harrington, D. (2002) Washing and chilling as critical control points in pork slaughter hazard analysis and critical control point (HACCP) systems. J. Appl. Microbiol. 92, 893-902.

14. Cheung, P.Y., Chan, C.W., Wong, W., Cheung, T.L., Kam, K.M. (2004) Evaluation of two real-time polymerase chain reaction pathogen detection kits for Salmonella spp. in food. Lett. Appl. Microbiol. 39(6), 509-515.

15. Cheung, P.Y., Kwok, K.K., Kam, K.M. (2007) Application of BAX system, Tecra UniqueTM Salmonella test, and a conventional culture method for the detection of Salmonella in ready-to-eat and raw foods. J. Appl. Microbiol. 103(1), 219-227.

16. Dorak, M.T. (2006) REAL-TIME PCR. In http ://www .dorak.iiifo/gcnctics/realtimc.html, Vol. 2006, Edited by M.T. Dorak.

17. Ellingson, J.L., Anderson, J.L., Carlson, S.A., Sharma, V.K. (2004) Twelve hour real-time PCR technique for the sensitive and specific detection of Salmonella in raw and ready-to-eat meat products. MoI. Cell. Probes 18(1), 51-57.

18. Hoorfar, J., Ahrens, P., Radstrόm, P. (2000) Automated 5' Nuclease PCR Assay for Identification of Salmonella enterica. J. Clin. Microbiol. 38(9), 3429-3435.

19. Hoorfar, J., Cook, N., Malorny, B., Wagner, M., De Medici, D., Abdulmawjood, A., Fach, P. (2003) Making Internal Amplification Control Mandatory for Diagnostic PCR. J. Clin.

Microbiol. 41(12), 5835.

20. Hoorfar, J., Malorny, B., Abdulmawjood, A., Cook, N., Wagner, M., Fach, P. (2004) Practical considerations in design of internal amplification controls for diagnostic PCR assays. J. Clin. Microbiol. 42(5), 1863-1868. 21. Julio, S.M., Heithoff, D.M., Mahan, MJ. (2000) ssrA (tmRNA) plays a role in Salmonella enterica serovar Typhimurium pathogenesis. J. Bacteriol. 182(6), 1558-1563.

22. Keiler, K.C., Shapiro, L., Williams, K.P. (2000) tmRNAs that encode proteolysis-inducing tags are found in all known bacterial genomes: A two-piece tmRNA functions in Caulobacter. Proceedings of the National Academy of Sciences 97(14), 7778-7783.

23. Lay, M.J., Wittwer, CT. (1997) Real-time fluorescence genotyping of factor V Leiden during rapid-cycle PCR. Clin. Chem. 43(12), 2262-2267.

24. Lofstrom, C, Knutsson, R., Axelsson, C. E., Radstrom, P. (2004) Rapid and Specific Detection of Salmonella spp. in Animal Feed Samples by PCR after Culture Enrichment. Appl. Environ. Microbiol. 70(1), 69-75.

25. Malorny, B., Paccassoni, E., Fach, P., Bunge, C, Martin, A., Helmuth, R. (2004) Diagnostic real-time PCR for detection of Salmonella in food. Appl. Environ. Microbiol. 70(12), 7046-7052.

26. Malorny, B., Tassios, P.T., Radstrom, P., Cook, N., Wagner, M., Hoorfar, J. (2003) Standardization of diagnostic PCR for the detection of foodborne pathogens. International Journal of Food Microbiology 83(1), 39-48.

27. Moore, M.M., Feist, M.D. (2007) Real-time PCR method for Salmonella spp. targeting the stn gene. J. Appl. Microbiol. 102(2), 516-530.

28. Moore, S.D., Sauer, R.T. (2007) The tmRNA System for Translational Surveillance and Ribosome Rescue. Annual Review of Biochemistry 76(1), 101-124. 29. O' Grady, J., Sedano-Balbas, S., Maher, M., Smith, T., Barry, T. (2008) Rapid real-time PCR detection of Listeria monocytogenes in enriched food samples based on the ssrA gene, a novel diagnostic target. Food Microbiol. 25(1), 75-84.

30. Parkhill, J., Dougan, G., James, K.D., Thomson, N.R., Pickard, D., Wain, J., Churcher, C, Mungall, KX. , et al. (2001) Complete genome sequence of a multiple drug resistant Salmonella enterica serovar Typhi CT18. Nature 413(6858), 848-852.

31. Roche (2007) Colour Compensation for Hydroloysis Probe Assays. Technical Note No. LC 21/2007, 1-5.

32. Rodriguez-Lazaro, D., Hernandez, M. (2006) Molecular Methodology in Food Microbiology Diagnostics: Trends and Current Challenges. In IUFoST World Congress, Vol. DOI: 10.1051/IUFoST:20060643, pp. 1085-1099. IUFoST.

33. Rosenstraus, M., Wang, Z., Chang, S.Y., DeBonville, D., Spadoro, J.P. (1998) An internal control for routine diagnostic PCR: design, properties, and effect on clinical performance. J. Clin. Microbiol. 36(1), 191-197. 34. Rossen, L., Norskov, P., Holmstrom, K., Rasmussen, O.F. (1992) Inhibition of PCR by components of food samples, microbial diagnostic assays and DNA-extraction solutions. Int. J. Food Microbiol. 17(1), 37-45.

35. Rybicki, E. (2001) PCR Primer Design and Reaction Optimisation. In Molecular Biology Techniques Manual, Vol. 2001Edited by V.E. Coyne, M.D. James, S.J. Reid, E.P. Rybicki. Cape Town: www.mcb.uct.ac.za/pcroptim.htm.

36. Siebert, P.D., Larrick, J.W. (1992) Competitive PCR. Nature 359(6395), 557-558.

Table 1 Salmonella and non-Salmonella serovars/strains used in real-time based assay design and validation Salmonella ^'Non-Salmonella

Serovar Source Organism Source

Agona NCTC 11377^a Escherichia coli ATCC 25922° Anatum SARB 2^b Escherichia coli NCTC 09001³ Braenderup NCTC 05750^a Escherichia coli NDC 544^f Bredeney NCTC 0573 T Citrobacter freundi NCTC 09750^a Derby SARB l l^b Citrobacter freundi NCTC 8090^a Dublin NCTC 09676^a Citrobacter diversus CCFRA7119^g Enteritidis ATCC 13076° Citrobacter koseri NCTC 10768^a Enteritidis PT4 NCTC 13349^a Enterobacter cloacae NCTC 11933^a Gallinarum NCTC 423,287/91^a Enterobacter agglomerans NCTC 09381³ Gallinarum NCTC 13346^a Enterobacter intermedins NDC 427^f Goldcoast NSRL^C L Enterobacter aerogens NCTC 10006^a Hadar ULjub MI 2° Enterobacter sakazaki NCTC 11467^a Heidelberg NCTC 5717^a Enterococcus faecium ATCC 35667° Infantis Uljub ^Λ VΕ 35/94^C Enterococcus faecalis NCTC 12697^a Kentucky NCTC 05799^a Bacillus cereus NCTC 07464^a Livingstone NCTC 09125^a Klebsiella oxyctoca ATCC 43086° London NCTC 05777^a Klebsiella pneumoniae ATCC 13883° Manhattan NCTC 06245^a Pseudomonas aeruginosa NCTC 12903^a Newport SARB 36^b Lactobacillus plantarum ATCC 8014° Nottingham NCTC 07832^a Pseudomonas putida ATCC 49128° Panama SARB 40^b Pseudomonas fragi DSM 3456^h Saint-Paul Uljub VF S-13/95^c Staphylococcus haemolyticus ATCC 29970° Senftenburg SARB 59^b Staphylococcus epidermidis AFRC¹ Stanley SARB 60^b Staphylococcus saprophyticus ATCC 15305° Typhimurium ATCC 14028° Streptococcus lactis NCDO 2003^J Typhimurium LT 2 NCTC 12416^a Aeromonas hydrophila ATCC 35654° Typhimurium DT 104 NCTC 13348^a Arthrobacter globiformis ATCC 8010° Uganda NCTC 06015^a Leuconostoc mesenteroides ATCC 8293° Virchow NCTC 05742^a Acinetobacter calcoaceticus ATCC 23055° Gaminara NCTC 5797^a Proteus mirabilis DSM 4479^h a NCTC National Collection of Type Cultures; SARB Salmonella Reference Collection B, University College Cork, Ireland; ^c ATCC American Type Culture Collection; ^d NSRL National Salmonella Reference Laboratory, University College Hospital Galway, Ireland; ^e ULjub University of Ljubljana, Slovenia; ^f NDC National Diagnostic Centre, NUI Galway, Ireland; ^g CCFRA Campden and Chorleywood Food Research Association; ^b DSM German Collection of Microorganisms and Cell Cultures; ' AFRC Ashtown Food Research Centre, Teagasc, Dublin, Ireland; ^J NCDO National Collection of Dairy Organisms c/o NCIMB Ltd., Aberdeen, Scotland, United Kingdom. Table 2 Sequences of primers and probes used in design and development of the ssrA Salmonella assay

Name Sequence Size (bp) Tm (⁰C) ^{■ '}

FPF" 5 ' -CCTCGTAAAAAGCCGCA-3 ' 17 59.1 FPR^b 5 ' -GAGTTGAACCCGCGTC-3 ' 16 59.0

Entero-tm F^b 5 ' -GGGGCTGATTCTGGATTCGA-S ' 20 63.6 Entero-tm R^b 5'- TGGTGGAGCTGGCGGGA-S' 17 67.5 SAM 1^C FAM-CAAACGACGAAACCTACGCTTTAGC-BBQ 25 66.8 SAM 2^C FAM-AGACTAGCCTGATTCGTTTTAACGCT-BBQ 26 66.6 IAC-Entero^b ROX-TCAAACCCAAAAGAGATCGCGTGGA-BHQ2 2 233 68.9 ^aMelting temperatures (Tm) were calculated using MeltCalc software (www.meltcalc.de) bMWG Biotech, Anzingerstr. 7a, 85560 Ebersberg, Germany

⁰TIB MOLBIOL GmbH, Eresburgstrasse 22-23, D-12103 Berlin, Germany

Table 6 Ct values recorded for cDNA, DNA and RNA templates using the modified enrichment / real-time PCR and RT-PCR methods.

Template Ct value 20 cDNA prepared from total RNA un-treated with DNase 8.70 cDNA prepared from DNase treated RNA 11.64

DNA 17.12

Total RNA un-treated with DNase 19.61 DNase treated total RNA H₂O neg

Table 3 Blind study samples - inoculation data and combined enrichment / real-time PCR

results

Swab inoculated with:

Swab no. ^'Non-Salmonella (CFU/ml) Salmonella (CFU/ml) Results

Swab 1 E. coli (-10) Typ. DT104 (-100), Derby (-10)

Swab 2 E. cloacae (~10) Typ. (-10), Dublin (-10)

Swab 3 Citro. (-1,000) Living. (-100), Typ. DT104 (-10)

Swab 4 E. coli (-100) N/A

Swab 5 E. coli (-10) Typ. (-10), Typ. DT104 (-1)

Swab 6 Citro. (-100) Derby (-10), Typ. (-100)

Swab 7 E. coli (-10), E. cloacae (-10) Dublin (-10)

Swab 8 E. coli (-10) Derby (-10), Living. (-10)

Swab 9 Blank Blank

Swab 10 E. cloacae (-10) Typ. (-10), Typ. DT104 (-100)

Swab 11 Citro. (-100) Derby (-10), Living. (-1)

Swab 12 Citro. (-100) Typ. DT104 (-10)

Swab 13 Citro. (-100) Derby (-10)

Swab 14 E. cloacae (-100) N/A

Swab 15 £. co/z (-10) Typ. (-10)

Swab 16 Citro. (-100) Dublin (-10)

Swab 17 Czϊro. (-100) Living. (-100)

Swab 18 Citro. (-100), £. co/z (-10) Typ. (-10)

Swab 19 is. cloacae (-10) Derby (-1), Dublin (-100)

Swab 20 Czϊro. (-100) N/A

Swab 21 N/A Typ. DT104 (-10), Typ. (-10), Living. (-10)

Swab 22 £. co/z (-10) Derby (-1), Typ. (-1)

Swab 23 £. c/oαcαe (-100) Typ. DT104 (-10), Living. (-10)

Swab 24 Czϊro. (-100) Derby (-10), Typ. (-1), Living. (-10)

Derby = 5". Derby SARB 11 ; Dublin = 5". Dublin NCTC 09676; Living. = 5". Livingstone NCTC 09125; Typ. DT104 = 5". Typhimurium DT 104 NCTC 13348; Typ. = 5". Typhimurium ATCC 14028; E. coli = E. coli NCTC 09001 ; E. cloacae = Enterobacter cloacae NCTC 10005; Citro. = Citrobacter freundi NCTC 09750. Table 4 Salmonella and non-Salmonella serovars / strains used in the design, development and validation of a modified enrichment / real-time RT-PCR method.

Salmonella ^'Non-Salmonella

Serovar Strain Organism Strain

Agona NCTC 11377^a Acinetobacter calcoaceticus ATCC 23055°

Anatum SARB 2^b Aeromonas hydrophila ATCC 35654°

Braenderup NCTC 05750^a Arthrobacter globiformis ATCC 8010°

Bredeney NCTC 05731³ Bacillus cereus NCTC 07464^a

Derby SARB l l^b Citrobacter diversus CCFRA7119^s

Dublin NCTC 09676^a Citrobacter freundii NCTC 8090^a

Enteritidis ATCC 13076° Citrobacter freundii NCTC 09750^a

Enteritidis PT4 NCTC 13349^a Citrobacter koseri NCTC 10768^a

Gallinarum NCTC 423,287/91^a Enterobacter aerogenes NCTC 10006^a

Gallinarum NCTC 13346^a Enterobacter agglomerans NCTC 09381 ^a

Goldcoast NSRL^d Enterobacter cloacae NCTC 11933^a

Hadar ULjub MI 2° Enterobacter intermedins NDC 427

Heidelberg NCTC 5717^a Enterobacter sakazaki NCTC 11467^a

Infantis Uljub VF 35/94^c Enterococcus faecalis NCTC 12697^a

Kentucky NCTC 05799^a Enterococcus faecium ATCC 35667°

Livingstone NCTC 09125^a Escherichia coli ATCC 25922°

London NCTC 05777^a Escherichia coli NDC 544^f

Manhattan NCTC 06245^a Escherichia coli NCTC 09001 ^a

Newport SARB 36^b Klebsiella oxytoca ATCC 43086°

Nottingham NCTC 07832^a Klebsiella pneumoniae ATCC 13883°

Panama SARB 40^b Lactobacillus plantarum ATCC 8014°

Saint-Paul Uljub VF S-13/95^c Leuconostoc mesenteroides ATCC 8293°

Senftenberg SARB 59^b Proteus mirabilis DSM 4479^h

Stanley SARB 60^b Pseudomonas aeruginosa NCTC 12903^a

Typhimurium ATCC 14028° Pseudomonas fragi DSM 3456^h

Typhimurium LT 2 NCTC 12416^a Pseudomonas putida ATCC 49128°

Typhimurium DT 104 NCTC 13348^a Staphylococcus epidermidis AFRC¹

Uganda NCTC 06015^a Staphylococcus haemolyticus ATCC 29970°

Virchow NCTC 05742^a Staphylococcus saprophyticus ATCC 15305°

Gaminara NCTC 5797^a Streptococcus lactis NCDO 2003^J Table 5 Ct values recorded for 30 Salmonella strains using the modified enrichment / real-time PCR and RT-PCR methods - Phase 1 Validation

Serovar Strain PCR (Ct values) RT-PCR (Ct values)

S. Agona NCTC 11377 17.01 7.58

S. Anatum UCC SARB 2 16.50 6.87

S. Braenderup NCTC 05750 17.25 7.82

S. Bredeney NCTC 05731 18.99 7.82

S. Derby UCC SARB I l 17.04 8.40

S. Dublin NCTC 09676 16.07 6.77

S. Enteritidis ATCC 13076 16.32 6.98

S. Enteritidis PT4 NCTC 13349 16.18 6.18

S. Gallinarum NCTC 423,287/91 27.73 24.00

S. Gallinarum NCTC 13346 18.39 9.92

S. Goldcoast NSRL 17.62 7.70

S. Hadar ULjub MI 2 17.61 7.53

S. Heidelberg NCTC 5717 17.31 5.84

S. Infantis ULjub VF 35/94 17.51 6.88

S. Kentucky NCTC 05799 17.19 7.15

S. Livingstone NCTC 09125 16.12 6.82

S. London NCTC 05777 17.09 6.09

S. Manhattan NCTC 06245 17.41 8.07

S. Newport UCC SARB 36 16.05 5.96

S. Nottingham NCTC 07832 17.86 8.93

S. Panama UCC SARB 40 17.64 9.90

S. Saintpaul ULjub VF S- 13/95 18.23 6.73

S. Senftenberg UCC SARB 59 16.65 6.20

S. Stanley UCC SARB 60 16.37 6.32

S. Typhimurium ATCC 14028 17.05 5.80

S. Typhimurium LT2 NCTC 12416 17.80 5.89

S. Typhimurium DTl 04 NCTC 13348 16.60 6.78

S. Uganda NCTC 06015 18.36 7.71

S. Virchow NCTC 05742 17.17 8.63

S. Gaminara NCTC 5797 17.05 8.73 Table 7: Non-Salmonella organisms used in real-time PCR assay validation

Organism Source Organism Source

Aeromonas hydrophila DSM^a 30015 Hafnia alvei DSM^a 30163 Alcaligenes faecalis DSM^a 30030 Helicobacter pylori DSM^a 21031 Anaerococcus hydrogenalis DSM^a 7454 Lactobacillus acidophilus DSM^a 20079 Bacillus cereus DSM^a 31 Lactobacillus fermentum DSM^a 20055 Bacillus subtilis DSM^a 704 Morganella morganii DSM^a 30164 Bacteroides fragilis DSM^a 2151 Mycoplasma genitalium NCTC^d 10195 Bacteroides thetaiotaomicron DSM^a 2079 Neisseria gonorrhoeae DSM^a 9188 Bifidobacterium breve DSM^a 20213 Proteus vulgaris DSM^a 2140 Candida albicans CBS^b 2700 Providencia stuartii DSM^a 4539 Candida glabrata CBS^b 138 Pseudomonas aeruginosa DSM^a 50071 Candida krusei CBS^b 573 Pseudomonas putida DSM^a 291 Candida parapsilosis ATCC^C 96137 Rahnella aquatilis DSM^a 4594 Candida tropicalis CBS^b 94 Saccharomyces cerevisiae DSM^a 7837 Citrobacter amalonaticus DSM^a 4593 Serratia liquefaciens DSM^a 4487 Citrobacter freundii DSM^a 30039 Serratia marcescens DSM^a 1608 Corynebacterium genitalium NCTC^d 12451 Serratia odoriferae DSM^a 4582 Corynebacterium genitalium NCTC^d 11859 Serratia rubidaea DSM^a 4480 Edwardsiella tarda DSM^a 30052 Shigella boydii DSM^a 7532 Enterobacter cloacae ATCC^C 13047 Shigella flexneri DSM^a 4782 Enterococcus faecalis DSM^a 20371 Shigella sonnei DSM^a 5570 Enterococcus faecium DSM^a 20477 Staphylococcus aureus NCTC^d 11965 Escherichia hermannii DSM^a 4560 Staphylococcus epidermidis DSM^a 20044 Ewingella americana DSM^a 4580 Stenotrophomonas maltophilia DSM^a 50170 Fusobacterium nucleatum DSM^a 15643 Streptococcus agalactiae BCGVf 15081 Fusobacterium varium DSM^a 19868 Streptococcus bovis DSM^a 20480

Streptococcus equinus DSM^a 20558

Vibrio parahaemolyticus DSM^a 10027

Yersinia enterocolitica DSM^a 4780

^aDSM German Collection of Microorganisms and Cell Cultures; ^bCBS Centraalbureau Voor Schimmelcultures; ⁰ATTC American Type Culture Collection; ^dNCTC National Collection of Type Cultures; ⁶BCCM Belgian Co-ordinated Collections of Micro-organisms.

Claims

Claims

1. A method for the detection and identification of Salmonella comprising:- (a) taking a sample from an object to be tested, (b) carrying out at least one culture enrichment step on the sample,

(c) isolating nucleic acid from the sample,

(d) contacting the isolated nucleic acid with at least one oligonucleotide probe derived from the ssrA gene,

(e) hybridizing under high stringency conditions any nucleic acid that may be present in the sample with the oligonucleotide to form a probe :target duplex; and

(f) determining whether a probe :target duplex is present; the presence of the duplex positively identifying the presence of Salmonella in the test sample.

2. A method as claimed in claim 1 wherein the culture enrichment step in carried out in Buffered Peptone Water or Rappaport Vasilliadis Soya Broth.

3. A method as claimed in claim 2 wherein the method comprises carrying out a first culture enrichment step in Buffered Peptone Water, and carrying out a second culture enrichment step in Rappaport Vasilliadis Soya Broth.

4. A method as claimed in any preceding claim wherein the oligonucleotide probe is selected from :- CAAACGACGAAACCTACGCTTTGC, and

AGCGTTAAAACGAATCAGGCTAGTCT and sequences substantially homologous thereto or substantially complimentary thereto, which also have a function in diagnostics based on the ssrA gene.

5. A method as claimed in claim 4 wherein the test sample is contacted with both oligonucleotide probes.

6. A method as claimed in any of claims 2 to 5 wherein the enrichment step in Buffered Peptone Water is carried out for 16 to 20 hours.

7. A method as claimed in claim 6 wherein the enrichment step is carried out at 36 to 38⁰C.

8. A method as claimed in any of claims 2 to 7 wherein the enrichment step in Rappaport Vasilliadis Soya Broth is carried out for 4 to 8 hours.

9. A method as claimed in claim 8 wherein the enrichment step is carried out at 41 to 43⁰C.

10. A method as claimed in any preceding claim wherein an internal amplification control which is amplified with the same primer set as the Salmonella target, is used in step (f).

11. A method as claimed in claim 10 wherein the internal amplification control is an E. coli. ssrA gene fragment.

12. A method as claimed in any preceding claim wherein nucleic acid is isolated from the culture supernatant of the sample.

13. A method as claimed in claim 12 wherein the method also comprises a step for releasing nucleic acid from any cells of the target organism that may be present in said test sample.

14. A method as claimed in any preceding claim wherein the nucleic acid isolated is DNA.

15. A method as claimed in any preceding claim wherein the nucleic acid isolated is RNA which is reverse transcribed to produce cDNA, prior to hybridisation.

16. .A diagnostic kit for detection and identification of Salmonella species, comprising probes based on the ssrA gene.

17. A kit as claimed in claim 16 comprising a probe selected from :- CAAACGACGAAACCTACGCTTTGC, and AGCGTTAAAACGAATCAGGCTAGTCT and sequences substantially homologous thereto, or substantially complementary to a portion thereof and having a function in diagnostics based on the ssrA gene.

18. A diagnostic kit as claimed in claim 17 further comprising a primer for amplification of at least a portion of the ssrA gene.

19. A diagnostic kit as claimed in claim 17 or 18 further comprising a forward and a reverse primer for a portion of the ssrA gene.

20. A nucleic acid molecule selected from the group consisting of:-

CAAACGACGAAACCTACGCTTTGC, and

AGCGTTAAAACGAATCAGGCTAGTCT and sequences substantially homologous thereto, or substantially complementary to a portion thereof and having a function in diagnostics based on the ssrA gene.

21. A method substantially as described herein with reference to the accompanying drawings.

22. A diagnostic kit substantially as described herein with reference to the accompanying drawings.