WO2010142702A1 - Méthode de détection, en temps réel, d'espèces de salmonella basée sur les acides nucléiques, rapide - Google Patents

Méthode de détection, en temps réel, d'espèces de salmonella basée sur les acides nucléiques, rapide Download PDF

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WO2010142702A1
WO2010142702A1 PCT/EP2010/058039 EP2010058039W WO2010142702A1 WO 2010142702 A1 WO2010142702 A1 WO 2010142702A1 EP 2010058039 W EP2010058039 W EP 2010058039W WO 2010142702 A1 WO2010142702 A1 WO 2010142702A1
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salmonella
nucleic acid
nctc
sample
detection
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Thomas Barry
Justin O Grady
Sheila Mcguinnesss
Terry James Smith
Majella Maher
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National University of Ireland Galway NUI
National University of Ireland
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National University of Ireland Galway NUI
National University of Ireland
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • 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.
  • 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.
  • GTP Good hygiene practice
  • HACCP Hazard Analysis Critical Control Point
  • 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].
  • 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].
  • 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.
  • target nucleic acid 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).
  • 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 NaH 2 PO 4 H 2 O and 1.85 g/1 EDTA, ph adjusted to 7.4 with NaOH), 0.5% SDS, 5xDenhardt's reagent and lOO ⁇ g/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 NaH 2 PO 4 H 2 O and 1.85 g/1 EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS, 5xDenhardt's reagent and 100 ⁇ g/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 NaH 2 PO 4 H 2 O 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 ⁇ g/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.
  • stringency is achieved by applying temperature conditions and ionic buffer conditions that are particular to that in-vitro amplification technology.
  • probeency 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.
  • 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%.
  • “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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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 :-
  • the culture enrichment step in carried out in Buffered Peptone Water or Rappaport
  • 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 :-
  • test sample is contacted with both oligonucleotide probes.
  • 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 0 C and preferably 37 0 C.
  • 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 0 C and preferably
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • Salmonella serovar S. Agona
  • selected closely related species S. Agona
  • 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 100cm 2 in the Salmonella real-time
  • Salmonella serovars were incubated at 37 0 C while non-Salmonella organisms were incubated at 30 0 C or 37 0 C, as appropriate.
  • 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
  • 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.
  • the modified culture enrichment method involved primary enrichment in BPW (100 ml) for 18 h at 37 0 C. After incubation, 100 ⁇ l of pre-enrichment culture was transferred to 10 ml RVS and incubated at 42 0 C 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 0 C until use.
  • PBS phosphate buffered saline
  • 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 0 C 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 Wales, 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
  • 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 ⁇ M and working stocks of 5 ⁇ M and 2 ⁇ M respectively, were prepared. All stocks were stored at -20 0 C.
  • Salmonella real-time PCR assay Real-time PCR reactions were performed on the LightCycler® 2.0 Instrument (Roche
  • PCR was performed in a final volume of 20 ⁇ l including 2 ⁇ l DNA template in LightCycler hybridisation buffer with MgCl 2 adjusted to 5 mM concentration.
  • LightCycler® Uracil-DNA Glycosylase (Roche Diagnostics) (0.3 ⁇ l) was also added to each reaction to minimise risk of contamination.
  • PCR primers, FPF and FPR (0.5 ⁇ M final concentration) and TaqMan probes,
  • SAM 1, SAM 2 and IAC -Entero (0.2 ⁇ M final concentration) were added to the reaction mixture.
  • 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 95 0 C and an annealing for 30 sec at 64°C.
  • 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.
  • Fresh carcass swabs were collected in local abattoirs.
  • Four sites on the carcass were swabbed (4 x 100 cm 2 ) 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).
  • 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 ⁇ l. 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
  • RNA samples were DNase treated using the 'TURBO DNA-free Kit' (Ambiors) before use in real-time RT-PCR.
  • Whole samples i.e. 50 ⁇ l
  • 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 0 C 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)
  • 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).
  • BPW buffered peptone water
  • 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.
  • 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.
  • 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 10 6 to 10° genome equivalents. Analysis was performed in triplicate for 3 Salmonella serovars, each with a different probe binding combination i.e. S.
  • 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 cm 2 , and 100% when spiked with 10 CFU/100 cm 2 and above (Fig. 3). Ninety five percent confidence intervals of 91.1% - 98.3% were observed. Blind sample study
  • 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.
  • 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.
  • 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.
  • 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.
  • a positive signal from a secondary target 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
  • 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.
  • 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.
  • 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.
  • salts Magnetic chloride (anhydrous) 13.58 g/L
  • Malachite green 0.036 g/L residual blue colouring
  • 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 cm 2 , and 100% when spiked with 10 CFU/100 cm 2 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 cm 2 in the presence of naturally occurring background flora can be performed in 26 hours.
  • 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.
  • 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
  • Swab 19 is. cloacae (-10) Derby (-1), Dublin (-100)
  • Organism Source Organism Source of

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Abstract

L'invention concerne un procédé rapide et précis pour détecter des espèces de Salmonella. Ledit procédé comprend une application particulière pour détecter des Salmonella dans des produits alimentaires, mais aussi des applications dans d'autres domaines, telles que des applications médicales et d'autres applications de diagnostics. Le dit procédé implique une méthode combinée d'enrichissement/PCR en temps réel pour détecter rapidement des Salmonella sur de la viande fraîche en fonction du gène ssrA.
PCT/EP2010/058039 2009-06-08 2010-06-08 Méthode de détection, en temps réel, d'espèces de salmonella basée sur les acides nucléiques, rapide Ceased WO2010142702A1 (fr)

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* Cited by examiner, † Cited by third party
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WO2012095714A1 (fr) * 2011-01-13 2012-07-19 Bigtec Private Limited Sondes et amorces pour la détection de la typhoïde
WO2016079304A1 (fr) * 2015-02-17 2016-05-26 Danmarks Tekniske Universitet Procédé rapide de détection de salmonella dans la viande
CN106676164A (zh) * 2016-09-07 2017-05-17 上海出入境检验检疫局动植物与食品检验检疫技术中心 一种在待测样品中检测菠萝成分的方法
CN119662510A (zh) * 2025-02-24 2025-03-21 深圳市虹彩新材料科技有限公司 一种用于聚酯塑料降解的微生物酶制剂及其制备方法

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* Cited by examiner, † Cited by third party
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WO2012095714A1 (fr) * 2011-01-13 2012-07-19 Bigtec Private Limited Sondes et amorces pour la détection de la typhoïde
WO2016079304A1 (fr) * 2015-02-17 2016-05-26 Danmarks Tekniske Universitet Procédé rapide de détection de salmonella dans la viande
CN106676164A (zh) * 2016-09-07 2017-05-17 上海出入境检验检疫局动植物与食品检验检疫技术中心 一种在待测样品中检测菠萝成分的方法
CN119662510A (zh) * 2025-02-24 2025-03-21 深圳市虹彩新材料科技有限公司 一种用于聚酯塑料降解的微生物酶制剂及其制备方法

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