Real-time PCR for the detection of Salmonella spp. in food: An alternative approach to a conventional PCR system suggested by the FOOD-PCR project

A real-time PCR assay using non-patented primers and a TaqMan probe for the detection and quantification of Salmonella spp. is presented. The assay is based on an internationally validated conventional PCR system, which was suggested as a standard method for the detection of Salmonella spp. in the FOOD-PCR project. The assay was sensitive and specific. Consistent detection of 9.5 genome equivalents per PCR reaction was achieved, whereas samples containing an average of 0.95 genome equivalents per reaction were inconsistently positive. The assay performed equally well as a commercially available real-time PCR assay and allowed sensitive detection of Salmonella spp. in artificially contaminated food. After enrichment for 16 h in buffered peptone water (BPW) or universal pre-enrichment broth (UPB) 2.5 CFU/25 g salmon and minced meat, and 5 CFU/25 g chicken meat and 25 ml raw milk were detected. Enrichment in BPW yielded higher numbers of CFU/ml than UPB for all matrices tested. However, the productivity of UPB was sufficient, as all samples were positive with both real-time PCR methods, including those containing less than 300 CFU/ml enrichment broth (enrichment of 5 CFU/25 ml raw milk in UPB).     

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.     

Rapid detection of low levels of Listeria in foods and next-day confirmation of L. monocytogenes

Outbreaks of foodborne listeriosis caused by Listeria monocytogenes in recent years, and the high mortality rate associated with listeriosis, have raised the need for reliable and rapid detection of the pathogen. A simple, automated method was developed for the detection of Listeria organisms in foods. It consists of a 6-h pre-enrichment step followed by overnight incubation in selective broth at 35 degrees C. Changes in light transmittance in the selective broth are registered continuously by an optical sensor of the BioSys instrument (MicroSys, Ann Arbor, MI), and recorded in the computer. Esculin hydrolysis by listeriae results in black coloration of the media that causes a sharp drop in light transmittance, whereas negative samples remain colorless. Confirmation of L. monocytogenes is carried out only on esculin-positive samples and is completed within 6 h. Detection of 10-50 cells of Listeria inoculated into 25 g of food was confirmed in shell eggs, milk and ground beef. Naturally contaminated raw and ready-to-eat foods were further screened to validate the procedure.     

Detection of salmonellas in confectionery products by conductance

A modified lysine decarboxylase broth has been developed which could be used with a Bactometer M123 to differentiate salmonellas from other bacteria by the characteristics of the conductance detection curve. The medium was used in combination with a selenite cystine trimethylamine oxide dulcitol medium to screen 50 strains of salmonellas and 42 strains of other organisms to establish detection curve magnitude and rate values which could be used to identify curves specific to salmonellas. The combination of media detected all salmonellas tested except Salmonella pullorum. The two media were used to screen 100 inoculated product samples with the Bactometer instrument, in parallel with traditional plating procedures, and using various combinations of pre-enrichment and selective enrichment incubation periods. After 24 h pre-enrichment, the Bactometer system detected more positive samples than the conventional plating procedures after pre-enrichment and selective enrichment. It is considered that these media used in parallel in the Bactometer after conventional pre-enrichment could provide a 48 h screening procedure for salmonellas with a sensitivity comparable to present plating procedures.     

Detection of salmonellas in confectionery products by conductance

A modified lysine decarboxylase broth has been developed which could be used with a Bactometer M123 to differentiate salmonellas from other bacteria by the characteristics of the conductance detection curve. The medium was used in combination with a selenite cystine trimethylamine oxide dulcitol medium to screen 50 strains of salmonellas and 42 strains of other organisms to establish detection curve magnitude and rate values which could be used to identify curves specific to salmonellas. The combination of media detected all salmonellas tested except Salmonella pullorum . The two media were used to screen 100 inoculated product samples with the Bactometer instrument, in parallel with traditional plating procedures, and using various combinations of pre-enrichment and selective enrichment incubation periods. After 24 h pre-enrichment, the Bactometer system detected more positive samples than the conventional plating procedures after pre-enrichment and selective enrichment. It is considered that these media used in parallel in the Bactometer after conventional pre-enrichment could provide a 48 h screening procedure for salmonellas with a sensitivity comparable to present plating procedures.     

Evaluation of different DNA extraction procedures for the detection of Salmonella from chicken products by polymerase chain reaction

Polymerase chain reaction (PCR) was used after a short pre-enrichment culture to detect Salmonella subspecies in chicken fillets. A direct PCR assay performed with chicken meat inoculated with Salmonella Typhimurium produced no PCR products. Six different DNA extraction protocols were tested to recover efficiently Salmonella DNA after a short incubation period. Three of them gave results that were reliable, rapid and sensitive. Successful protocols used Proteinase K and/or a centrifugation step to concentrate the samples. For reliable detection of Salmonella subspecies, a few thousand bacterial cells per ml must be present. To obtain this number of bacterial cells with an inoculation of about one cell in 25 g of ionized food products, it was necessary to incubate samples for at least 10 h before PCR. A larger inoculum of approximately 10 cells in 25 g of ionized food products, required 8 h in culture broth to give positive results by PCR-based assay.     

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.     

Improved bioluminescent enzyme immunoassay for the rapid detection of Salmonella in chicken meat samples

AIMS: To evaluate an improved bioluminescent enzyme immunoassay (BEIA) using biotinylated firefly luciferase for the rapid detection of Salmonella in naturally contaminated chicken meat samples. METHODS AND RESULTS: Capture agents and lipopolysaccharide (LPS) extraction reagents for Salmonella were investigated to improve the sensitivity of the BEIA. Also, the use of Oxoid SPRINT (Simple Pre-enrichment and Rapid Isolation New Technology) as a pre-enrichment and selective medium for 26-h BEIA detection of Salmonella in chicken meat samples was examined. The use of polymyxin B as a capture agent on solid support and 3-[(3-Cholamidopropyl) dimethylammonio] propanesulfonic acid (CHAPS) for extraction of the LPS facilitated sensitive detection of Salmonella. Of 120 chicken meat samples, 25 samples were positive using the improved BEIA with the SPRINT and 25 samples were positive using the SPRINT followed by the standard isolation methods. CONCLUSIONS: The improved BEIA, in which polymxin B was used as a capture agent and CHAPS was used for extraction of the antigen, had a sensitivity of 96.0% and a specificity of 98.9% for the detection of Salmonella in chicken meat. SIGNIFICANCE AND IMPACT OF THE STUDY: The improved BEIA combined with the SPRINT medium for the detection of Salmonella in chicken meat samples produced comparable results to the culture methods in 26 h.     

Detection of Salmonella spp. in food by a rapid PCR-hybridization procedure.

A rapid and sensitive PCR-hybridization procedure for detection of Salmonella serovars in food samples was developed. This method is based on three subsequent steps: (1) extraction of nucleic acids from a 2 ml aliquot of the pre-enrichment medium used for the conventional culture method after 6 h of incubation at 37 degrees C; (2) amplification with primers selected from the sequences of invE and invA genes; (3) Southern blot and hybridization with a biotin labeled oligonucleotide probe. The entire procedure requires 30 h. The PCR-hybridization assay was able to detect as little as 50 fg of purified chromosomal DNA of S. typhimurium and 0.2 cfu g-1 of an artificially contaminated food sample. Of 245 food samples analyzed by culture and PCR-hybridization, 20 were positive by both methods and 16 were positive by PCR-hybridization only. None of the 209 PCR-negative samples tested positive by culture. The sensitivity, specificity, alpha and beta error values of the results of the PCR-hybridization procedure, compared with those of culture, were 100, 92.9, 0 and 7.1%, respectively. These results indicate that a short pre-enrichment and PCR-hybridization could be used as a screening test for the detection of Salmonella in food samples.     

Eight-hour PCR-based procedure for the detection of Salmonella in raw oysters

The aim of this study was to evaluate the ability of a nested PCR system to detect Salmonella senftenberg in raw oysters. The specific primers of the PCR were derived from the invA gene sequence, essential for Salmonella invasiveness into epithelial cells. First, for the extraction of DNA, four methods (guanidine isothiocyanate, E.Z.N.A. Mollusc Kit, Chelex-100, and lysis with detergents) were compared. A nested PCR method combined with 3.5 h pre-enrichment in buffered peptone water (BPW) and DNA extraction by the resin Chelex-100 is proposed for the detection of S. senftenberg in oyster samples. The detection limit of the method is less than 0.1 CFU/ml (<1 CFU/g of oyster). This procedure is shown to be an excellent tool for the sensitive detection of S. senftenberg from naturally contaminated oysters, with results being obtained within 8 h.     

Salmonella-TEK, a rapid screening method for Salmonella species in food.

A micro-enzyme-linked immunosorbent assay (micro-ELISA) using the Salmonella-TEK screen kit was tested for the detection of Salmonella spp. in pure cultures as well as in 30 artificially contaminated food samples and in 45 naturally contaminated food samples. Different raw, fleshy foods and processed foods were used as test products. The artificially contaminated minced meat samples were preenriched in buffered peptone water, and after incubation, different selective enrichment broths were tested. The micro-ELISA optical density values after enrichment and isolation of the different broths were very analogous. The quickest method to detect Salmonella spp. in different foods is to enrich them with Salmosyst broth, which reduces the total analysis time to 31 h. The Salmonella-TEK kit for Salmonella spp. provides a promising test for the detection of Salmonella antigens in food even when they are present at a low concentration (1 to 5 CFU/25 g). The cross-reaction of the anti-Salmonella antibodies, especially to other gram-negative bacteria, is nil.     

Efficiency of different enrichment and isolation procedures for the detection of Salmonella serotypes in edible offal

Rapid detection systems for Salmonella in foodstuffs are currently being developed. However, existing standards still call for application of traditional methods employing pre-enrichment followed by selective enrichment and isolation. The efficacy of various methods was tested using 264 chicken and lamb organ meats. Pre-enrichment was carried out in Tryptone Soy Broth (TSB) and enrichment in Tetrathionate Brilliant Green Broth (TTB) at 37°C, Selenite Broth with Brilliant Green and Sulphapyridine at 37°C and 43°C, and Rappaport-Vassiliadis Broth (RV 10) at 42°C. The isolation media were Brilliant Green Agar (BGA), Deoxycholate Citrate Agar, Hektoen Enteric Agar (HEA) and Salmonella-Shigella Agar.
Enrichment in RV/42°C followed by isolation on BGA as recommended by ISO standard no. 6579 and enrichment in TTB/37°C followed by isolation in HEA, no longer recommended by that standard, produced the best results. Low percentages of positive samples and difficulties in detecting Salmonella are the result of interference by competing organisms (Enterobacteriaceae) and the number of salmonellas present after enrichment.
A total of 528 samples (TSB, eggs, lamb liver and chicken liver) were inoculated with Salm. enteritidis, Salm. kapemba and Salm. virchow , and the preceding experiment was repeated. All the TSB and egg samples tested positive, but the percentage of positive samples from the lamb and chicken liver was only 81–92%. Recovery of the salmonellas did not depend upon the method employed or the serotype inoculated but instead on interference by competing flora and the numbers of Salmonella present in the samples.     

Detection of Salmonella enterica serovar Typhi (S. Typhi) by selective amplification of invA, viaB, fliC-d and prt genes by polymerase chain reaction in mutiplex format

AIMS: Development of a PCR assay that can target multiple genes for rapid detection of Salmonella enterica serovar Typhi (S. Typhi) from water and food samples. METHODS AND RESULTS: PCR primers for invasion, O, H and Vi antigen genes, invA, prt, fliC-d and viaB were designed and used for the rapid detection of S. Typhi by multiplex PCR. Internal amplification control, which co-amplified with prt primers, was also included in the assay. The results showed that all cultures of Salmonella were accurately identified by the assay with no nonspecific amplification in other cultures. The assay had 100% detection probability when a cell suspension of 10(4) CFU ml(-1) (500 CFU per reaction) was used. Salmonella Typhi bacteria were artificially inoculated in the water and food (milk and meat rinse) samples and detected by mPCR after overnight pre-enrichment in buffered peptone water. No Salmonella bacteria could be detected from water samples collected from the field by mPCR or standard culture method. CONCLUSIONS: The developed mPCR assay provides specific detection of S. Typhi. SIGNIFICANCE AND IMPACT OF THE STUDY: Rapid methods for detection of S. Typhi from complex environmental matrices are almost nonexistent. The mPCR assay reported in this study can be useful to identify S. Typhi bacteria in field environmental samples.     

Improvement in Salmonella detection in milk and dairy products: comparison between the ISO method and the Oxoid SPRINT Salmonella test

The Oxoid SPRINT Salmonella test was compared with the ISO method (ISO 6579: 1993) for the detection of Salmonella in milk and dairy products. Samples were artificially contaminated, in some cases with sublethally injured salmonellas. Experiments with raw milk, soft cheese made from heat-treated milk (mould-ripened and with smear) and soft cheese with smear made from raw milk showed no significant differences between the SPRINT and ISO methods. With dried milk products and mould-ripened soft cheese made from raw milk the reference method gave significantly more positive results. The addition of ferrioxamine E to pre-enrichment (ISO) or pre-enrichment/enrichment broth (SPRINT test) did not improve Salmonella detection.     

Rapid detection of salmonellae in poultry with the magnetic immuno-polymerase chain reaction assay.

Rapid detection of salmonellae in chicken meat was accomplished by using the magnetic immuno-polymerase chain reaction assay (MIPA). A direct polymerase chain reaction assay performed with chicken meat spiked with Salmonella typhimurium resulted in poor sensitivity (approximately 10(7) CFU/g of meat). The use of immunoseparation with a Salmonella serogroup B-specific monoclonal antibody improved the sensitivity, but enrichment was required for the detection of low levels of contamination. Enrichment for 6 h in either buffered peptone water, lactose broth containing tergitol-7, or selenite-cystine broth resulted in the detection of an initial inoculum of 100 CFU per g of meat. Enrichment of the salmonellae present on 25 g of spiked chicken meat for 24 h in either buffered peptone water or selenite-cystine broth before detection by the MIPA yielded a detection limit of approximately 0.1 CFU/g of meat. A detection limit of approximately 1 CFU/g of meat was obtained when the spiked meat was stored at -20 degrees C before enrichment for 24 h and analysis with the MIPA. Although the MIPA was developed for S. typhimurium, a MIPA in which a panel of six monoclonal antibodies specific for Salmonella serogroups A through E was used detected the presence of 0.1 CFU of Salmonella enteritidis per g of chicken meat. These data indicate that the method is applicable to other commonly isolated serotypes.     

Salmonella-TEK, a rapid screening method for Salmonella species in food

A micro-enzyme-linked immunosorbent assay (micro-ELISA) using the Salmonella-TEK screen kit was tested for the detection of Salmonella spp. in pure cultures as well as in 30 artificially contaminated food samples and in 45 naturally contaminated food samples. Different raw, fleshy foods and processed foods were used as test products. The artificially contaminated minced meat samples were preenriched in buffered peptone water, and after incubation, different selective enrichment broths were tested. The micro-ELISA optical density values after enrichment and isolation of the different broths were very analogous. The quickest method to detect Salmonella spp. in different foods is to enrich them with Salmosyst broth, which reduces the total analysis time to 31 h. The Salmonella-TEK kit for Salmonella spp. provides a promising test for the detection of Salmonella antigens in food even when they are present at a low concentration (1 to 5 CFU/25 g). The cross-reaction of the anti-Salmonella antibodies, especially to other gram-negative bacteria, is nil.     

Increased recovery of salmonellae from environmental samples enriched with buffered peptone water.

The incidence and persistence of salmonellae in weather pools on the top of Stone Mountain were investigated with lactose and buffered peptone water used as pre-enrichment broths. A total of 162 samples were collected from 16 weather pools over a 3-month period. The use of buffered peptone water increased the recovery of salmonellae by approximately 25%. The combined use of direct enrichment in tetrathionate broth containing brilliant green dye and pre-enrichment in buffered peptone water followed by enrichment in tetrathionate broth made it possible to detect all 37 of the contaminated samples. All of the isolates were Salmonella bareilly, the only serotype recovered in a previous study. All but one of the isolations were made from moist or wet samples. S. bareilly was isolated from rabbit dung and litter collected near the weather pools, but attempts to trap rabbits for study were unsuccessful. Random samples taken along a side of the mountain yielded S. bareilly in weather pools within the upper third of the mountain; below this level, S. weslaco and S. memphis were recovered, but not S. bareilly.     

免疫磁珠富集技术联合选择性培养基快速检测单增李斯特菌

单增李斯特菌是一种危害极大的食源性致病菌,建立快速及特异的检测方法对于食品安全监控尤为重要。文中联合免疫磁珠与选择性培养基对不同浓度(101~105CFU/mL)单增李斯特菌进行检测,并对3种李斯特菌、金黄色葡萄球菌及副溶血弧菌进行交叉试验;同时模拟食物污染,探索免疫磁珠-平板法检测样品的检测限以及该方法的最快检测时间。结果显示特异性免疫磁珠联合选择性平板法可检出浓度为103CFU/mL及以上的单增李斯特菌;牛奶样品仅需6 h增菌能被检出,检测限为0.7 CFU/mL。联合使用免疫磁珠富集技术与选择性培养基,能在30 h内完成对牛奶样品的检测,较国标法减少38 h以上,且具有同等的灵敏度。     

Increased recovery of salmonellae from environmental samples enriched with buffered peptone water.

The incidence and persistence of salmonellae in weather pools on the top of Stone Mountain were investigated with lactose and buffered peptone water used as pre-enrichment broths. A total of 162 samples were collected from 16 weather pools over a 3-month period. The use of buffered peptone water increased the recovery of salmonellae by approximately 25%. The combined use of direct enrichment in tetrathionate broth containing brilliant green dye and pre-enrichment in buffered peptone water followed by enrichment in tetrathionate broth made it possible to detect all 37 of the contaminated samples. All of the isolates were Salmonella bareilly, the only serotype recovered in a previous study. All but one of the isolations were made from moist or wet samples. S. bareilly was isolated from rabbit dung and litter collected near the weather pools, but attempts to trap rabbits for study were unsuccessful. Random samples taken along a side of the mountain yielded S. bareilly in weather pools within the upper third of the mountain; below this level, S. weslaco and S. memphis were recovered, but not S. bareilly.     

Evaluation of new culture media for rapid detection and isolation of salmonellae in foods.

Conventional methods for Salmonella detection in foods can require up to 6 and at least 4 days. We have observed that the total analysis time can be reduced to 48 h by using Salmosyst broth as a liquid medium for both preenrichment and selective enrichment and Rambach agar (RA), a new selective plate medium. In samples of artificially contaminated ground beef Salmonella enteritidis was detected at a concentration of 0.4 CFU/g (10 CFU/25 g) by both a conventional method and the new method. Of 519 samples of foods for sale, 38 were Salmonella positive by both methods while 471 were negative. Nine samples which were negative by the conventional method were positive by the Salmosyst-RA method, while one sample positive by the first method was negative by the last. Therefore, the Salmosyst-RA method showed 97.9% sensitivity compared with the 81.2% sensitivity of the conventional method. The new method was also highly specific (98% specificity) in presumptive identification of Salmonella colonies. Furthermore, a 6-h preenrichment in Salmosyst broth has been proved sufficient for the repair of heat-injured Salmonella cells and for subsequent recovery by selective enrichment. In conclusion, the Salmosyst-RA method shows several advantages over both conventional and rapid noncultural methods: (i) only two media are required instead of the five media for conventional methods; (ii) in real time it is comparable to other rapid noncultural methods, which require 30 to 31 h; (iii) it is highly sensitive and specific; and (iv) it allows the isolation of Salmonella strains which can be characterized by appropriate phenotypic and genotypic typing methods for epidemiological investigations.