Application of magnetic immuno-polymerase chain reaction assay for detection of <Emphasis Type="Italic">Salmonella</Emphasis> spp. in chicken meats

Since contaminated chicken meats have been the principal foodborne source of the contamination of Salmonella to human beings and cultural detection methods are labor-intensive and time-consuming, a study evaluating the performance of the combination of two techniques that are immunomagnetic separation (IMS) and polymerase chain reaction (PCR) for the detection of Salmonella in chicken meats was conducted. The IMS and PCR assay combines selective extraction of Salmonella by specific antibodies with primer-specific (primer pair based on the sequence of invA gene) PCR amplification. Initially chicken meat samples, in which no Salmonella contamination had been determined by using ISO 6579 reference method, were inoculated with Salmonella Enteritidis culture and subsequently the shortest non-selective pre-enrichment time, that had been needed for the detection of approximately 1 or 10 CFU/mL chicken meat levels of target bacteria by magnetic immuno-PCR assay, was found by using 14, 12, 10 and 8-h periods. In conclusion, it was found that magnetic immuno-PCR assay was able to detect 1–10 CFU Salmonella/25 g chicken meat, after only incorporating a non-selective pre-enrichment period of 12 h. Therefore, an overall 16-h (magnetic immuno-PCR assay in conjunction with 12-h non-selective pre-enrichment) magnetic immuno-PCR assay statistically evaluated as sufficient (p = 0.182 > 0.05) for rapid and sensitive detection of approximately 1–10 CFU Salmonella from 25 g chicken meat samples. Accordingly, 16-h magnetic immuno-PCR assay can be promising for routine use in the detection of Salmonella in chicken meat samples, and it consequently may prevent the risk of Salmonella infections in regard to chicken meats.     

Multiplex Real-time PCR for the Simultaneous Detection of Salmonella spp. and Listeria monocytogenes in Food Samples

In this study, we have developed a rapid method for the simultaneous detection of Listeria monocytogenes and Salmonella spp. in foods, combining culture enrichment and a multiplex real-time polymerase chain reaction (PCR). The assay used two pre-existing primer-probe sets, labelled with different reporter dyes to enable the direct distinction of the original contaminating agent. Amplification efficiency and inclusivity/exclusivity of the combined assay was successfully assessed. The overall process included the culture enrichment based on the ISO standard, consisting of 24 h incubation in appropriate media (Half Fraser Broth for Listeria and buffered peptone water (BPW) for Salmonella), followed by a single DNA extraction of mixed enrichment aliquots, and real-time PCR detection of the hly and bipA genes of L. monocytogenes and Salmonella spp., respectively. An internal amplification control, co-amplified during the PCR run, was included in the assay to verify the results. The tool was evaluated with a variety of artificially inoculated samples of fresh products and ready to eat and cooked dishes, allowing the identification of the target pathogens down to 5 CFU/25 g of food sample. Moreover, the analysis saved a considerable amount of time compared to the ISO standard, being performed in less than 2 working days. Specificity, sensitivity and accuracy were satisfactorily tested by comparison to the standard methods ISO 11290-2:1998 and ISO 6579:2002, suggesting that the tool has a great potential as a reliable alternative for food safety assurance providing rapid detection of both pathogens in food samples.     

A New Real-Time PCR Assay for the Specific Detection of Salmonella spp. Targeting the bipA Gene

A straightforward real-time polymerase chain reaction (PCR)-based assay was designed and evaluated for the detection of Salmonella spp. in food and water samples. This new assay is based on the specific detection of the bipA gene of Salmonella, which encodes a protein of the guanosine triphosphate (GTP)-binding elongation family that displays global modulating properties, by regulating a wide variety of downstream processes. The new method correctly identified all 48 Salmonella strains used in the inclusivity test, and did not detect all 30 non-Salmonella species tested. The method was evaluated by analyzing 120 diverse food and water samples enriched in buffered peptone water. The bipA-based real-time PCR assay showed 100% efficiency, sensitivity, and specificity compared to the invA-based method previously published, which was developed as a part of a European project for the standardization of PCR methods in food microbiology. The assay includes an independent internal amplification control (IAC) in each reaction to control false negative results.     

Multiplex PCR for the simultaneous detection of Salmonella spp. and Salmonella Enteritidis in food

Multiplex PCR assay (mPCR) for the detection of Salmonella spp. and S. Enteritidis was developed in this study using artificially contaminated chicken carcasses. The assay showed 100% specificity to detect approximately 1 CFU of Salmonella in 10 g of chicken skin after non‐selective enrichment. The mPCR was evaluated in Minas cheese, fresh pork sausage and chicken carcasses commercially available. Salmonella spp. was detected in nine of sixty‐six chicken carcasses, five of fifty‐two cheese samples, and five of fifty‐two sausage samples. The serovar Enteritidis was detected in two samples of contaminated sausage. The mPCR results were confirmed by conventional culture and biochemical identification of the isolates. Serotyping confirmed the presence of S. Enteritidis in sausage samples and showed contamination by serovars Schwarzengrund and Montevideo in chicken carcasses.     

Validation of a Duplex Real-Time PCR for the Detection of Salmonella spp. in Different Food Products

A 5′ nuclease duplex real-time polymerase chain reaction (PCR) assay was developed and validated with various food products for the specific and fast detection of Salmonella spp. in food. The assay used previously published primers in combination with a newly developed probe targeting the invA gene. An internal amplification control, which is coamplified in a duplex PCR, was included in the assay. The analysis of 1,934 natural food samples with real-time PCR and the cultural method in parallel resulted in a relative accuracy of 100% and 99.84% respectively, depending on the enrichment procedure in which buffered peptone water and selective enrichment in Rappaport–Vassiliadis (RV) broth were employed. The duplex real-time PCR assay has proven to be a specific, sensitive and fast screening method for Salmonella spp. in food. The overall analysis time of the PCR method was approximately 28 h, in contrast to 4 to 5 days with conventional Salmonella diagnostics. The developed assay has been shown to be a reliable diagnostic tool for use in routine analysis. It has been validated thoroughly and has become an official method in Germany for the detection of Salmonella spp. in food.     

A New Validated Real-Time PCR-Based Method for the Specific and Fast Detection of Cronobacter spp. in Infant Formula

In this study, a real-time polymerase chain reaction (PCR)-based method was designed for the fast detection of Cronobacter spp. (a newly proposed genus formerly known as Enterobacter sakazakii) in infant formula. The real-time PCR was positively tested with 70 Cronobacter strains, including members of all the species of this genus, and 88 non-Cronobacter strains. This new PCR-based system was validated against the reference standard ISO/TS 22964: 2006 (ISO International Organization for Standardization 2006) using 70 food matrices including powdered infant formula, follow-up formula, and hydrolyzed cereals for infants. The detection limit of the technique was found to be of 1 cfu in 10 g of food, fulfilling the requirements of the European Commission. The time of analysis, which comprises around 3–6 days using the reference method, is considerably reduced to less than 24 h using the real-time PCR-based system hereby described, allowing food industry a faster release of the stocks for commercialization. Moreover, this method includes an internal amplification control, co-amplified during each PCR run to verify the results.     

Improving the enrichment procedure for Enterobacteriaceae detection

The current ISO standard method for detection of Enterobacteriaceae (21528-1:2004) includes enrichment in EE broth which has been shown to be inhibitory to some members of this family, notably Cronobacter spp. A shortened procedure omitting the EE broth has been proposed, however competition from Gram-positive flora may be detrimental to the effective recovery of low levels of target organisms in some sample matrices. In this study we investigated novel cost effective modifications, designed to improve ISO 21528-1:2004 for the detection of Enterobacteriaceae. Initial experiments used a worse-case scenario involving stressed Enterobacteriaceae strains known to grow poorly in laboratory media as well as representative background competitors from powdered milk. The interaction between the Enterobacteriaceae and their competitors was characterised and additives to enhance the growth of target strains over non-target strains were investigated.Supplementation of BPW with 40 μM 8-hydroxyquinoline, 0.5 g L⁻¹ ammonium iron(III) citrate, 0.1 g L⁻¹ sodium deoxycholate and 0.1 g L⁻¹ sodium pyruvate (BPW-S) improved the recovery of Enterobacteriaceae from artificially and naturally contaminated samples. This improvement of the pre-enrichment broth may also be of interest for methods designed to detect specific foodborne pathogens belonging to the Enterobacteriaceae (e.g. Salmonella spp., Cronobacter spp.) that require a pre-enrichment step in BPW.     

Development of multiplex real-time PCR with Internal amplification control for simultaneous detection of Salmonella and Cronobacter in powdered infant formula

Contamination of powdered infant formula (PIF) by the bacteria Cronobacter spp. and Salmonella enterica was deemed a matter of great concern by the World Health Organization and the Food and Agriculture Organization of the United Nations in 2004. Therefore, we developed a rapid and sensitive multiplex real-time PCR assay for the simultaneous detection of Cronobacter and Salmonella in PIF. In addition, an internal amplification control (IAC) was also included for exclusion of false negative results in this study. The quantitative detection range for pure cultures in this optimized multiplex real-time PCR assay was 10³ to 10⁸ CFU/ml for both Salmonella and Cronobacter. When our established multiplex real-time PCR system was applied to artificially contaminated PIF, the detection limit was 10³ CFU/ml for Salmonella and Cronobacter without enrichment. The commercial PIF was then inoculated with Salmonella and Cronobacter at 10, 1 and 0.1 CFU per gram of formula and the single enrichment broth samples were analyzed by multiplex real-time PCR after enrichment for 9, 12, and 24 h. At 12 h post-enrichment, we could detect Salmonella and Cronobacter at initial inoculation levels of approximately 0.1 CFU/g in PIF. Additionally, stable fluorescent IAC signals could be assessed between 29 and 34 cycles of PCR amplification. Results from this study showed that the multiplex real-time PCR assay is an effective method for the rapid and simultaneous detection and quantification of Cronobacter and Salmonella in PIF.     

Development of a real-time PCR assay for detection and quantification of enterotoxigenic members of Bacillus cereus group in food samples

A highly sensitive real-time PCR (qPCR) procedure, targeting the phosphatidylcholine-specific phospholipase C gene (pc-plc), was developed for specific detection and quantification of strains belonging to Bacillus cereus group. The target region was selected based on the enterotoxigenic profiles of 75 Bacillus strains. The inclusivity and exclusivity of the RTi-PCR assay were assessed with 59 isolates of the B. cereus group, 16 other Bacillus spp., and 4 non-Bacillus strains. The assay was also used to construct calibration curves for different food matrices, and it had a wide quantification range of 6 log units using both serial dilutions of purified DNA and calibrated cell suspensions of B. cereus CECT 148^T. The detection limit for B. cereus in artificially contaminated liquid egg and reconstituted infant formula was about 3 CFU per reaction or 60 CFU/ml of food, with a relative accuracy of 86.27% to 116.12% in artificially contaminated liquid egg. Naturally contaminated food samples were tested for the presence of B. cereus with the standard method, a conventional PCR and the new developed RTi-PCR assay. Results showed that the new developed RTi-PCR assay is very suitable for detection and quantification of strains of B. cereus group in food samples without an enrichment step.     

Development and validation of a rapid real-time PCR based method for the specific detection of Salmonella on fresh meat

In this study, a combined enrichment/real-time PCR method for the rapid detection of Salmonella on fresh meat carcasses, was designed, developed and validated in-house following requirements outlined in ISO 16140:2003. The method included an 18 h non-selective enrichment in buffered peptone water (BPW) and a 6 h selective enrichment in Rappaport Vasilliadis Soya (RVS) broth, based on the traditional culture method, ISO 6579:2002. The real-time PCR assay included an internal amplification control (IAC), was 100% specific and was sensitive to one cell equivalent. The alternative method was validated against the traditional culture method and relative accuracy of 94.9%, sensitivity of 94.7% and specificity of 100% were determined using 150 fresh meat carcass swabs. This alternative method had a detection limit of 1–10 CFU/100 cm² for fresh meat carcass swabs and was performed in 26 h. Following further inter-laboratory studies, this alternative method could be suitable for implementation in testing laboratories for the analysis of carcass swabs.     

Multiplex TaqMan® detection of pathogenic and multi-drug resistant Salmonella

Overuse of antibiotics in the medical and animal industries is one of the major causes for the development of multi-drug-resistant (MDR) food pathogens that are often difficult to treat. In the past few years, higher incidences of outbreaks caused by MDR Salmonella have been increasingly documented. The objective of this study was to develop a rapid multiplex real-time polymerase chain reaction (PCR) assay for simultaneous detection of pathogenic and MDR Salmonella spp. A multiplex TaqMan®real-time PCR was designed by targeting the invasin virulence gene (invA), and four commonly found antibiotic resistance genes, viz. ampicillin, chloramphenicol, streptomycin and tetracycline. To avoid false negative results and to increase the reliability of the assay, an internal amplification control (IAC) was added which was detected using a locked nucleic acid (LNA) probe. In serially diluted (5 ng–50 fg) DNA samples, the assay was able to detect 100 genomic equivalents of Salmonella, while in a multiplex format, the sensitivity was 1000 genomic equivalents. The assay performed equally well on artificially contaminated samples of beef trim, ground beef of different fat contents (73:27, 80:20, 85:15 and 93:7), chicken rinse, ground chicken, ground turkey, egg, spinach and tomato. While the detection limit for un-enriched inoculated food samples was 10⁴ CFU/g, this was improved to 10 CFU/g after a 12-h enrichment in buffered peptone water, with 100% reproducibility. The multiplex real-time assay developed in this study can be used as a valuable tool to detect MDR virulent Salmonella, thus enhancing the safety of food.     

A real-time PCR for Detection of Pathogenic Yersinia enterocolitica in food combined with an Universal Internal Amplification Control System

Summary:  A real-time PCR system with an internal amplification control was developed for detection of pathogenic Yersinia) enterocolitica in food samples. The chromosomally encoded ail gene was chosen as PCR target. Sequences of plasmid pUC19 served as target for the internal amplification control. The method was validated in combination with sample enrichment in PSB and TSB broth using different food matrices spiked with Y. enterocolitica and naturally contaminated slaughterhouse samples. The results of the real-time PCR with internal control were verified by the cultural method according to EN ISO 10273:2003. The sensitivity of the real-time PCR with internal control is about 5 genome copies per reaction. Artificial contamination of food samples resulted in a detection level of 5 cfu per 25 g Y. enterocolitica in food samples. 100% of porcine tonsils and about 22% meat from pig heads were contaminated. The screening of samples by PCR prior to cultural analysis allows focusing on positive samples in routine analysis. This could result in a higher detection rate by cultural analysis.

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Zusammenfassung:  Für den Nachweis von pathogenen Yersinia enterocolitica wurde ein real-time PCR System mit interner Amplifikationskontrolle entwickelt. Das Nachweissystem für pathogene Y. enterocolitica basiert auf dem chromosomal kodierten ail-Gen. Als Zielsequenz der internen Amplifikationskontrolle dient eine Sequenz aus dem Plasmid pUC19. Zur Validierung der Methode wurden sowohl natürlich kontaminierte Proben aus einem Schlachthof als auch künstlich kontaminierte Proben verschiedener Lebensmittelmatrices verwendet. Die Anreicherung der Proben vor der molekularbiologischen Untersuchung erfolgte parallel in Tryptikase Soja-Bouillon (TSB) und in Pepton-Sorbit-Gallensalz-Bouillon (PSB). Die Ergebnisse der molekularbiologischen Untersuchungen wurden anschlie?end kulturell in Anlehnung an das Standardverfahren nach EN ISO 10273:2003 verifiziert. Die real-time PCR mit interner Amplifikationskontrolle weist eine Sensitivit?t von 5 Genomkopien pro Reaktionsansatz auf. Die Nachweisgrenze des Verfahrens, bestimmt anhand künstlich kontaminierter Proben, betr?gt etwa 5 KbE Y. enterocolitica pro 25 g Lebensmittel. Von den natürlich kontaminierten Proben aus einem Schlachthof waren die Tonsillen vom Schwein zu 100% mit pathogenen Y. enterocolitica kontaminiert, Schweinefleischabschnitte aus dem Kopfbereich wiesen einen Kontaminationsgrad von 22% auf. Ein Screening von Proben durch PCR erlaubt in der Routineanalytik die Fokussierung der kulturellen Analyse auf positive Proben. Dies k?nnte zu einer h?heren Nachweisrate durch das kulturelle Verfahren führen.

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Received: March 5. 2008; accepted: March 14. 2008     

A Rapid and Simple DNA Extraction Procedure to Detect Salmonella spp. and Listeria monocytogenes from Fresh Produce Using Real-time PCR

DNA isolation procedures significantly influence the outcome of PCR-based detection of human pathogens. Unlike clinical samples, DNA isolation from food samples, particularly from fresh and fresh-cut produce has remained a formidable task and has hampered the sensitivity and accuracy of molecular methods. We utilized a commercially available filter-based DNA isolation method (FTA) in conjunction with real-time PCR-based detection of Salmonella spp. and Listeria monocytogenes. The protocol uses filter paper discs impregnated with a patented chemical formulation that lyses cells, immobilizes DNA, and protects it from degradation. Use of the FTA method in conjunction with real-time PCR for the detection of Salmonella spp. and L. monocytogenes was compared with two commercially available DNA isolation procedures that are commonly used for high throughput real-time PCR pathogen detection systems. Both pathogens were successfully detected from artificially inoculated fresh and fresh-cut produce such as alfalfa sprouts, cilantro, green onion, broccoli, prepacked mixed salad, and spinach at low cell numbers (four to seven colony forming units per 25 g initial inoculum level before enrichment). The FTA protocol had distinct advantages of simplicity, biosafety, and compatibility for high throughput screening. This DNA preparation protocol was rapid, sensitive, required minimal handling, and reduced interference from produce-associated inhibitors of real-time PCR. Mention of brand names does not constitute an endorsement by the U.S. Department of Agriculture above others of a similar nature not mentioned.     

Effect of sample preparation and bacterial concentration on Salmonella enterica detection in poultry meat using culture methods and PCR assaying of preenrichment broths

We evaluated the sensitivity of a PCR assay in the detection of Salmonella enterica at the broth preenrichment step of poultry meat. A total of 162 retail poultry meat samples, which were prepared by manual massaging, stomacher or no homogenization were compared for Salmonella recovery. Using these homogenization methods, the PCR assay at the broth preenrichment step detected Salmonella in, respectively, 48.9%, 62.2% and 50.0% of meat and giblet samples detected as Salmonella-positive using the culture method. In ground chicken, however, Salmonella was detected in 21.7% of samples treated by stomacher homogenization, compared to 40.7% and 48% of untreated and hand-massaged samples, respectively. These results suggest that stomaching of ground chicken causes excessive effusion of food constituents, which affects PCR results. Using the most probable number (MPN) technique, Salmonella was detected at under 1.0 CFU/g in 12 ground chicken samples and under 10³ CFU/ml of broth in seven of the 12 broth-enriched samples, which considered the minimum concentration detectable by PCR assay. These results show that Salmonella detection using routine PCR assays is difficult in poultry meat, and in particular ground chicken, due to low amounts of Salmonella and the presence of inhibitors.     

Detection of Salmonella enteritidis and Salmonella typhimurium in foods using a rapid,multiplex real-time recombinase polymerase amplification assay

Salmonella has been recognized as a major foodborne pathogen for humans and animals. In this study, a multiplex real-time recombinase polymerase amplification (RPA) was developed for simultaneous detection of Salmonella enterica serovars, Salmonella enteritidis and Salmonella typhimurium, from chicken, eggs, lettuce, and papaya. The reaction was performed for 20 min at 35°C, and the detection limit of the assay was 10² CFU/ml for pure culture. In food application, the limit of detection (LOD) of S. enteritidis and S. typhimurium using multiplex real-time RPA without enrichment procedure was 10² CFU/25 g, respectively. After enrichment, the LOD of S. enteritidis and S. typhimurium was 10 CFU/25 g. Moreover, the result for Salmonella spp. was not significantly different from those obtained using a culture-based method. Additionally, the assay has a lower cross-reactivity with other pathogenic microorganisms and a good stability performance. Thus, the developed multiplex RPA assay could be used as a rapid tool for the detection of S. enteritidis and S. typhimurium in food.     

Development of rapid real-time PCR and most-probable-number real-time PCR assays to quantify enterotoxigenic strains of the species in the Bacillus cereus group

Members of the Bacillus cereus group may produce diarrheal enterotoxins and could be potential hazards if they enter the food chain. Therefore, a method capable of detecting all the species in the B. cereus group rather than B. cereus alone is important. We selected nhe as the target and developed a real-time PCR assay to quantify enterotoxigenic strains of the B. cereus group. The real-time PCR assay was evaluated with 60 B. cereus group strains and 28 others. The assay was also used to construct calibration curves for different food matrices and feces. The assay has an excellent quantification capacity, as proved by its linearity (R2 > 0.993), wide dynamic quantification range (10(2) to 10(7) CFU/g for cooked rice and chicken, 10(3) to 10(7) CFU/ml for milk, and 10(4) to 10(7) CFU/g for feces), and adequate relative accuracy (85.5 to 101.1%). For the low-level contaminations, a most-probable-number real-time PCR assay was developed that could detect as low as 10(0) CFU/ml. Both assays were tested with real food samples and shown to beconsiderably appropriate for B. cereus group detection and quantification.     

Prevalence and counts of Salmonella spp. in minimally processed vegetables in São Paulo, Brazil

Minimally processed vegetables (MPV) may be important vehicles of Salmonella spp. and cause disease. This study aimed at detecting and enumerating Salmonella spp. in MPV marketed in the city of São Paulo, Brazil. A total of 512 samples of MPV packages collected in retail stores were tested for Salmonella spp. and total coliforms and Escherichia coli as indication of the hygienic status. Salmonella spp. was detected in four samples, two using the detection method and two using the counting method, where the results were 8.8 × 10² CFU/g and 2.4 × 10² CFU/g. The serovars were Salmonella Typhimurium (three samples) and Salmonella enterica subsp. enterica O:47:z4,z23:- (one sample). Fourteen samples (2.7%) presented counts of E. coli above the maximum limit established by the Brazilian regulation for MPV (10² CFU/g). Therefore, tightened surveillance and effective intervention strategies are necessary in order to address consumers and governments concerns on safety of MPV.     

A real-time PCR for Detection of Pathogenic Yersinia enterocolitica in food combined with an Universal Internal Amplification Control System

A real-time PCR system with an internal amplification control was developed for detection of pathogenic Yersinia) enterocolitica in food samples. The chromosomally encoded ail gene was chosen as PCR target. Sequences of plasmid pUC19 served as target for the internal amplification control. The method was validated in combination with sample enrichment in PSB and TSB broth using different food matrices spiked with Y. enterocolitica and naturally contaminated slaughterhouse samples. The results of the real-time PCR with internal control were verified by the cultural method according to EN ISO 10273:2003. The sensitivity of the real-time PCR with internal control is about 5 genome copies per reaction. Artificial contamination of food samples resulted in a detection level of 5 cfu per 25 g Y. enterocolitica in food samples. 100% of porcine tonsils and about 22% meat from pig heads were contaminated. The screening of samples by PCR prior to cultural analysis allows focusing on positive samples in routine analysis. This could result in a higher detection rate by cultural analysis.