A real-time PCR for the detection of Salmonella Enteritidis in poultry meat and consumption eggs

A robust duplex 5' nuclease (TaqMan) real-time PCR was developed and in-house validated for the specific detection of Salmonella enterica subspecies enterica serovar Enteritidis in whole chicken carcass rinses and consumption eggs. The assay uses specifically designed primers and a TaqMan probe to target the Prot6e gene located on the S. Enteritidis specific 60-kb virulence plasmid. As an internal amplification control to monitor Salmonella DNA in the sample, a second primer/TaqMan probe set detects simultaneously the Salmonella specific invA gene. The assay identified correctly 95% of the 79 Salmonella Enteritidis strains tested comprising 19 different phage types. None of the 119 non-Enteritidis strains comprising 54 serovars was positive for the Prot6e gene. The assay detection probability was for 10(2) or more genome equivalents 100% and for 10 equivalents 83%. A pre-PCR sample preparation protocol including a pre-enrichment step in buffered peptone water, followed by DNA extraction was applied on low levels of artificially contaminated whole chicken carcass rinses and eggs from hens as well as 25 potentially naturally contaminated chickens. The detection limit was less than three CFU per 50 ml carcass rinse or 10 ml egg. The sensitivity and specificity compared to the traditional culture-based detection method and serotyping were both 100%. Twenty-five potentially naturally contaminated chickens were compared by the real-time PCR and the traditional cultural isolation method resulting in four Salmonella positive samples of which two were positive for the Prot6e gene and serotyped as S. Enteritidis. We show also that Salmonella isolates which have a rough lipopolysaccharide structure could be assigned to the serovar Enteritidis by the real-time PCR. This methodology can contribute to meet the need of fast identification and detection methods for use in monitoring and control measures programmes.     

Evaluation of real-time PCR vs automated ELISA and a conventional culture method using a semi-solid medium for detection of Salmonella

AIMS: Evaluation of iQ-Check PCR Salmonella for Salmonella detection in artificially and naturally contaminated food and environmental field samples. METHODS AND RESULTS: Artificially contaminated samples (poultry meat and ground red meat) subjected to cold- and freeze-stress, and 120 naturally contaminated samples (swabs and meat) were tested for Salmonella using the diagnostic semi-solid Salmonella medium (DIASALM) method, the Vidas assay and the iQ-Check PCR assay after 24 h enrichment in buffered peptone water. CONCLUSIONS: Both the iQ-Check PCR and the Vidas assay provide a rapid and user friendly screening method for detection of Salmonella. False negative samples were obtained for the inoculated samples using both the iQ-Check PCR assay and the Vidas method when Salmonella cells were severely stressed. In total 45 of 120 naturally contaminated field samples showed Salmonella positive using the DIASALM method. The agreement percentage with the DIASALM method was respectively 92% for the iQ-Check PCR and 95% for the Vidas method. SIGNIFICANCE AND IMPACT OF THE STUDY: False-negative samples were obtained for the inoculated samples using both the iQ-Check PCR assay and the Vidas method when Salmonella cells were severely stressed, e.g. freezing at -18 degrees C for 7 days. Of the 120 naturally contaminated field samples 45 showed Salmonella positive using the DIASALM method. The agreement percentage with the DIASALM method was 92% for the iQ-Check PCR and 95% for the Vidas method respectively.     

Development and application of a nested PCR to monitor brood stock salmonid ovarian fluid and spleen for detection of the fish pathogen Flavobacterium psychrophilum

AIMS: To develop a nested PCR to detect Flavobacterium psychrophilum based on the intergenic spacer region 16S-23S rRNA and in 16S rRNA for analysis of brood stock salmonid fish samples. METHODS AND RESULTS: The sensitivity and specificity of the test was evaluated using pure cultures, spiked and naturally contaminated samples. Samples were internal organs (spleen and kidney), eggs and ovarian fluid from rainbow trout and coho salmon from European fish farms (France, Spain). This nested PCR was more specific and sensitive that the nested PCR based on 16S rRNA sequences primers only. The detection limit of this PCR assay was one bacterium per PCR tube corresponding to 10 bacteria/mg of spleen and 5 bacteria/ml from ovarian fluid. Analysis of mixed ovarian fluid samples from reproductive salmonids in various French hatcheries demonstrated that 69% of hatcheries were contaminated with Fl. psychrophilum. The analysis of individual samples demonstrated that 39% of rainbow trout (Oncorhynchus mykiss) and 62.5% of coho salmon (O. kisutch) samples were contaminated. CONCLUSIONS: The results demonstrated a very sensitive and specific detection of this fish pathogen and that most of the female rainbow trout and coho salmon breeders analysed carry Fl. psychrophilum in the ovarian fluid. SIGNIFICANCE AND IMPACT OF THE STUDY: The understanding of Fl. psychrophilum dissemination and transmission and the detection of asymptomatic carriers is important for the development of free breeders stock and for significantly decreasing Flavobacteriose.     

Development and evaluation of a multiplex PCR for simultaneous detection of five foodborne pathogens

Aims: To develop a rapid multiplex PCR method for simultaneous detection of five major foodborne pathogens (Staphylococcus aureus, Listeria monocytogenes, Escherichia coli O157:H7, Salmonella Enteritidis and Shigella flexneri, respectively). Methods and Results: Amplification by PCR was optimized to obtain high efficiency. Sensitivity and specificity assays were investigated by testing different strains. With a multipathogen enrichment, multiplex PCR assay was able to simultaneously detect all of the five organisms in artificially contaminated pork samples. The developed method was further applied to retail meat samples, of which 80% were found to be positive for one or more of these five organisms. All the samples were confirmed by traditional culture methods for each individual species. Conclusions: This study reported a rapid multiplex PCR assay using five primers sets for detection of multiple pathogens. Higher consistency was obtained between the results of multiplex PCR and traditional culture methods. Significance and Impact of the Study: This work has developed a reliable, useful and cost‐effective multiplex PCR method. The assay performed equally as well as the traditional cultural method and facilitated the sensitive detection both in artificially contaminated and naturally contaminated samples.     

An alternative real-time PCR method to detect the Bacillus cereus group in naturally contaminated food gelatine: a comparison study

Aims:  Comparison of an internally controlled real-time PCR assay with the standard plate-based assay (ISO 21871) for the detection of Bacillus cereus group cells in gelatine.
Methods and Results:  A comprehensive TaqMan probe was designed allowing the real-time PCR assay to be fully inclusive and exclusive. An internal amplification control was designed and implemented at 500 copies per reaction without impact on target detection. Specific and selective detection of target cells was achieved with a quick and simple DNA preparation procedure. No significant difference (κ = 0·99) was observed between the performance of the real-time PCR and the standard plate-based method on naturally contaminated gelatines ( n  = 197). Relative accuracy, relative sensitivity and relative specificity were ≥99%.
Conclusions:  The real-time PCR assay is a valid alternative of the standard plate-based assay.
Significance and Impact of the Study:  The real-time PCR assay decreased the time between sample collection and result from 2 days to 2 h, while analysis cost did not increase. The gelatine-producing industry can ensure gelatine safety and quality in a much faster way.     

Detection and quantification of Aspergillus ochraceus in green coffee by PCR

AIMS: The aim of this study was to detect and quantify DNA of the ochratoxinogenic fungus Aspergillus ochraceus in green coffee and to compare the results with the ochratoxin A content of naturally contaminated samples. METHODS AND RESULTS: A DNA extraction protocol based on a combination of ultrasonification and a commercial kit was tested for the recovery of fungal DNA. PCR and real-time PCR protocols were established for the detection of A. ochraceus. Sensitivity of the PCR was checked by the addition of inoculated green coffee and pure fungal DNA to uncontaminated green coffee samples. The A. ochraceus DNA content of 30 naturally contaminated green coffee samples was determined and compared with the ochratoxin A concentrations. CONCLUSIONS: Aspergillus ochraceus can be rapidly and specifically detected in green coffee by PCR. A positive correlation between the ochratoxin A content and the DNA quantity was established. Significance and Impact of the Study: This work offers a quick alternative to the conventional mycological detection and quantification of A. ochraceus in green coffee.     

An alternative real-time PCR method for the detection of thermotolerant Bacillus sensu lato contaminants in naturally-contaminated gelatine

Aims:  Comparison of an internally-controlled real-time PCR assay with the current plate-based assay for the detection of Bacillus sensu lato contaminants in gelatine.
Methods and Results:  A comprehensive TaqMan^® probe was designed allowing the real-time PCR assay to be fully inclusive for the gelatine-contaminating Bacillus s.l. species. An internal amplification control was implemented at 500 copies per reaction without impact on target detection. Specific and selective detection of target cells was achieved with a quick and simple DNA preparation procedure. No significant difference (Kappa value = 0·94) was observed between the performance of the real-time PCR and the current plate-based method on naturally contaminated gelatines ( n  = 162). Relative accuracy, relative sensitivity and relative specificity were 97·5%.
Conclusions:  The real-time PCR assay is an adequate alternative of the current plate-based assay.
Significance and Impact of the Study:  The real-time PCR assay decreased the time between sample collection and result from 2 days to 2 h. The gelatine-producing industry can ensure gelatine quality in a much faster way.     

A real-time PCR assay for the detection of Campylobacter jejuni in foods after enrichment culture

A real-time PCR assay was developed for the quantitative detection of Campylobacter jejuni in foods after enrichment culture. The specificity of the assay for C. jejuni was demonstrated with a diverse range of Campylobacter species, related organisms, and unrelated genera. The assay had a linear range of quantification over six orders of magnitude, and the limit of detection was approximately 12 genome equivalents. The assay was used to detect C. jejuni in both naturally and artificially contaminated food samples. Ninety-seven foods, including raw poultry meat, offal, raw shellfish, and milk samples, were enriched in blood-free Campylobacter enrichment broth at 37 degrees C for 24 h, followed by 42 degrees C for 24 h. Enrichment cultures were subcultured to Campylobacter charcoal-cefoperazone-deoxycholate blood-free selective agar, and presumptive Campylobacter isolates were identified with phenotypic methods. DNA was extracted from enrichment cultures with a rapid lysis method and used as the template in the real-time PCR assay. A total of 66 samples were positive for C. jejuni by either method, with 57 samples positive for C. jejuni by subculture to selective agar medium and 63 samples positive in the real-time PCR assay. The results of both methods were concordant for 84 of the samples. The total time taken for detection from enrichment broth samples was approximately 3 h for the real-time PCR assay, with the results being available immediately at the end of PCR cycling, compared to 48 h for subculture to selective agar. This assay significantly reduces the total time taken for the detection of C. jejuni in foods and is an important model for other food-borne pathogens.     

Adding a selective enrichment step to the iQ-Check real-time PCR improves the detection of Salmonella in naturally contaminated retail turkey meat products

AIMS: The aim of this study was to compare the real-time iQ-Check Salmonella kit (Bio-Rad) with the immunocapture assay RapidCheck Salmonella method, and a conventional culture method (FSIS, USDA) in detecting Salmonella in naturally contaminated turkey meat products. This study was also designed to determine if a selective enrichment step might improve the real-time detection of Salmonella. METHODS AND RESULTS: Using the culture method, Salmonella was recovered from 49 out of 99 retail turkey meat samples collected. RapidCheck failed to detect 11 Salmonella samples that were positive by the culture method. The iQ-Check real-time PCR also failed to detect three samples that were positive by the culture method. However, when carried out after a selective enrichment step, the iQ-Check real-time PCR detected all 49 Salmonella samples recovered by the culture method. The iQ-Check real-time PCR detected the presence of Salmonella in some samples that were not recovered by the culture method. CONCLUSIONS: Adding a selective enrichment step to the iQ-Check real-time PCR improves the detection of Salmonella in naturally contaminated turkey meat samples. SIGNIFICANCE AND IMPACT OF THE STUDY: The iQ-Check Salmonella real-time PCR can be used as a rapid method to monitor Salmonella in turkey meat, together with conventional culture methodology.     

Direct detection by polymerase chain reaction (PCR) of Escherichia coli in water and soft cheese and identification of enterotoxigenic strains

PCR was used to develop a method to detect Escherichia coli in surface water and soft cheese, which does not require cultivation of bacteria. DNA sequences from the ma /B operon of E. coli were amplified to specifically detect this bacterium. Samples of surface water and soft cheese naturally contaminated with E. coli from less than 100 cells per g up to several times 10⁵cells per g were analysed by both the classical culture method and the PCR assay. Comparable results were obtained with both methods. Soft cheese samples artificially contaminated with various levels of enterotoxigenic E. coli were analysed with a second PCR test specific for the heat-labile enterotoxin type I (LTI) of E. coli. The detection limit was about 1000 bacteria per g of soft cheese. In addition, two soft cheese samples naturally contaminated with 2 times 10⁵and 6 times 10⁵ E. coli per g as determined by the culture method were analysed by LTI-PCR and found to contain low levels of enterotoxigenic E. coli.     

Detection of Magnaporthe grisea in infested rice seeds using polymerase chain reaction

AIM: To develop a diagnostic assay based on polymerase chain reaction for the detection of Magnaporthe grisea from infested rice seeds. METHODS AND RESULTS: Primers were designed based on the nucleotide sequence of the mif 23, an infection-specific gene of M. grisea. The primers amplified target DNA from genetically and geographically diverse isolates of the pathogen. The lowest concentration of template DNA that led to amplification was 20 rhog. No PCR product was detected when DNA from other fungi was used, indicating the specificity of the primers. With this PCR based seed assay, M. grisea was detected in rice seedlots with infestation rates as low as 0.2%. CONCLUSION: The PCR detection of M. grisea is simple, rapid, specific, sensitive and suitable for the routine detection of the pathogen in infested seeds. SIGNIFICANCE AND IMPACT OF THE STUDY: Introduction of the blast fungus into new areas where it has not been previously recorded could be avoided by the detection of infested seedlots. A PCR-based seed assay could facilitate risk assessment of naturally infested rice seeds; help design management programs and optimize fungicide use.     

A rapid, sensitive and automated method for detection of Salmonella species in foods using AG-9600 AmpliSensor Analyzer

The AG-9600 AmpliSensor Analyzer is an automated fluorescence-based system for detection of polymerase chain reaction (PCR) products. The principle of the AmpliSensor PCR assay involves amplification-mediated disruption of a fluorogenic DNA signal duplex (AmpliSensor) that is homologous to a target sequence within a 284-bp amplified fragment of the Salmonella invA gene. Since the assay is homogenous, the data can be obtained by direct measurement of fluorescence of the amplification mixture. The accumulation of the amplified product, reflected by the fluorescence index, is monitored cycle by cycle by the AG-9600 Analyzer. The detection limit of the assay was less than 2 colony-forming units (cfu) per PCR reaction using a pure culture of Salmonella typhimurium. In post-spiking experiments in which Salmonella was added to the overnight pre-enriched samples (chicken carcass rinses, ground beef, ground pork and raw milk), the detection limit of the assay was 2–6 cfu per PCR reaction. In pre-spiking experiments in which Salmonella was added to the samples prior to overnight pre-enrichment, the detection limit was less than 3 cfu per 25 g or 25 ml of food. The assay was up to 2 orders of magnitude more sensitive than detection by ethidium bromide-stained agarose gel electrophoresis. To further evaluate assay performance, 54 naturally contaminated chicken carcass rinses, 65 raw milk and six ground pork samples were tested in the study. Thirty-eight Salmonella- positive samples confirmed by the Modified Semi-solid Rappaport-Vassiliadis (MSRV) culture assay were found positive using the AmpliSensor assay. Two chicken carcass rinses found positive using the assay were MSRV-negative. In addition, relative quantification using the AmpliSensor assay was linear up to 3 logs of initial target concentration in artificially contaminated food samples.     

PCR detection of Listeria monocytogenes: a study of multiple factors affecting sensitivity

AIMS: To test, under comparable conditions, several parameters affecting sensitivity of PCR detection in order to establish a PCR procedure suitable for the routine detection of Listeria monocytogenes in food. METHODS AND RESULTS: Beef samples artificially inoculated were used to determine sensitivity of PCR detection under different parameters. As few as 1 CFU g(-1) were detected by DNA extraction using a DNeasy Tissue Kit (Qiagen GmhH, Hilden, Germany) of 1 ml aliquot and PCR amplification with primers directed to the hlyA gene. This PCR protocol was applied in 60 naturally contaminated foods, comparing two enrichment procedures with the traditional culture method. The highest number of positives was recorded by PCR following a 24-h pre-enrichment step at 30 degrees C and a 24-h enrichment step at 37 degrees C. Afterwards, it was applied in 217 naturally contaminated foods and 56 of them tested positive for L. monocytogenes in which only 17 tested positive using the culture method. CONCLUSIONS: The PCR procedure described has proved to be a rapid and sensitive method suitable for the routine analysis of different types of food. SIGNIFICANCE AND IMPACT OF THE STUDY: The method proposed for the detection of L. monocytogenes, has been validated in naturally contaminated food and is suitable to implement in the food industry.     

Evaluation of culture, ELISA and PCR assays for the detection of Salmonella in seafood

Aims: The study evaluated the efficiency of culture, enzyme‐linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) assays for the detection of Salmonella in naturally contaminated seafood. Methods and Results: In this study, 215 seafood samples comprising fish, shrimp, crab, clam, mussel, oyster, squid, cuttlefish and octopus from fish market of Cochin (India), were compared by culture, ELISA and PCR methods. Bacteriological Analytical Manual (BAM), U.S. Food and Drug Administration (USFDA) method was followed for culture assay, and Salmonella Tek, a commercial sandwich ELISA kit, was used for ELISA assay. Salmonella‐specific PCR assay was developed for 284 bp Salmonella‐specific invA gene amplicon. PCR assay exhibited 31·6% seafood positive for Salmonella followed by ELISA (23·7%) and culture method (21·3%). There was fair to excellent agreement between culture, ELISA and PCR assays (kappa coefficient values ranging from 0·385 to 1·0) for different seafood samples. Conclusion: The investigation revealed the greater concordance between culture and ELISA methods for seafood. Among the three methods, PCR assay was most sensitive. Lower detection rate with culture and ELISA assays could be attributed to greater sensitivity of the PCR method in the detection of Salmonella in seafood. Significance and Impact of the Study: We propose the incorporation of dual tests based on different principle and procedure for the routine analysis of Salmonella in seafood.     

Sequence of a cloned pR72H fragment and its use for detection of Vibrio parahaemolyticus in shellfish with the PCR.

The nucleotide sequence of pR72H cloned from Vibrio parahaemolyticus 93 was determined. We examined all V. parahaemolyticus gene sequences published in the GenBank-EMBL databases for homology and found that no other DNA sequence of V. parahaemolyticus was highly homologous to the sequence reported in this study. A pair of primers, VP33-VP32, derived from a pR72H fragment were selected to detect V. parahaemolyticus. The sensitivity of PCR detection for a pure culture of V. parahaemolyticus was 10 cells from crude bacterial lysates. Furthermore, a detection level of 2.6 fg, equivalent to 1 cell, was obtained by using purified chromosomal DNA as the template. The expected PCR products were obtained from all V. parahaemolyticus strains tested (n = 124), while no PCR amplicons were found in other vibrios or related genera (n = 50). High levels (10(6) to 10(10) CFU/ml) of Escherichia coli cells did not affect the PCR assay sensitivity. The presence of 10(8) V. parahaemolyticus cells or 10(9) E. coli cells in the PCR mixtures completely inhibited the PCR. When oyster samples were inoculated with V. parahaemolyticus 93 and cultured in tryptic soy broth containing 3% NaCl for 3 h at 35 degrees C, an initial sample inoculum level of 9.3 CFU/g was detected in a PCR assay with crude bacterial lysates. The PCR assay with enrichment culturing in salt polymyxin broth was compared with the conventional method for naturally contaminated shellfish and fish samples. We conclude that this PCR assay with enrichment culturing is a good alternative method for the detection of V. parahaemolyticus.     

Development of a PCR assay combined with a short enrichment culture for detection of Campylobacter jejuni in estuarine surface waters

Abstract Two extraction procedures were examined, and it was found that DNA recovered from Campylobacter jejuni lysed by the cetyltrimethylammonium bromide (CTAB) method was more suitable for use as a PCR template than DNA released by the boiling method. The region targeted for PCR amplification was a 1.73-kb portion of the flagellin A gene of C. jejuni . The detection limit was lower than 30 cells per 100 ml in artificially contaminated waters. PCR assay and conventional culturing method had the same sensitivity, but results of the PCR technique were available within 48 h and so shortened the time necessary for detection by 48 h.     

Comparison of PCR, Electrochemical Enzyme-Linked Immunosorbent Assays, and the Standard Culture Method for Detecting Salmonella in Meat Products

An electrochemical enzyme-linked immunosorbent assay (ELISA) coupled with flow injection analysis (ELISA-FIA) and a PCR-based method using ST11 and ST15 primers for detecting salmonellae in meat were evaluated in comparison with the International Organization for Standardization (ISO) culture method. The methods were applied to experimentally contaminated and naturally contaminated meat samples. The results showed that both ELISA-FIA and PCR allowed detection of salmonella in a product contaminated with a low number of the microorganisms (1 to 10 salmonellae/25 g) after only 5 h of incubation of preenrichment broth, and they were just as effective as the ISO method.     

Real-time PCR method for detection of pathogenic Yersinia enterocolitica in food

The current methods for the detection of pathogenic Yersinia enterocolitica bacteria in food are time consuming and inefficient. Therefore, we have developed and evaluated in-house a TaqMan probe-based real-time PCR method for the detection of this pathogen. The complete method comprises overnight enrichment, DNA extraction, and real-time PCR amplification. Also included in the method is an internal amplification control. The selected primer-probe set was designed to use a 163-bp amplicon from the chromosomally located gene ail (attachment and invasion locus). The selectivity of the PCR method was tested with a diverse range (n = 152) of related and unrelated strains, and no false-negative or false-positive PCR results were obtained. The sensitivity of the PCR amplification was 85 fg purified genomic DNA, equivalent to 10 cells per PCR tube. Following the enrichment of 10 g of various food samples (milk, minced beef, cold-smoked sausage, fish, and carrots), the sensitivity ranged from 0.5 to 55 CFU Y. enterocolitica. Good precision, robustness, and efficiency of the PCR amplification were also established. In addition, the method was tested on naturally contaminated food; in all, 18 out of 125 samples were positive for the ail gene. Since no conventional culture method could be used as a reference method, the PCR products amplified from these samples were positively verified by using conventional PCR and sequencing of the amplicons. A rapid and specific real-time PCR method for the detection of pathogenic Y. enterocolitica bacteria in food, as presented here, provides a superior alternative to the currently available detection methods and makes it possible to identify the foods at risk for Y. enterocolitica contamination.     

PCR-based assay for the detection of Curtobacterium flaccumfaciens pv. flaccumfaciens in bean seeds

AIMS: To develop a PCR-based protocol for the rapid, sensitive and specific detection of Curtobacterium flaccumfaciens pv. flaccumfaciens (Cff) in bean seeds. METHODS AND RESULTS: A pair of PCR primers (CffFOR2-CffREV4), targeting the sequence of a cloned DNA fragment of 550 bp amplified in Repetitive-sequence-based-PCR (Rep-PCR) experiments, were designed and shown to specifically amplify a 306-bp DNA fragment using Cff DNA as template. Moreover, this PCR protocol was demonstrated to successfully detect Cff in naturally infected bean seeds in 36 h. CONCLUSIONS: A specific, highly sensitive and rapid PCR assay for the detection of Cff was achieved. SIGNIFICANCE AND IMPACT OF THE STUDY:Cff is a seed-borne bacterium on the EPPO A2 quarantine list; this procedure may be useful for routine diagnosis of Cff, overcoming the problems of conventional techniques.