Salmonella real-time PCR-Nachweis

Salmonella belongs to the most important bacterial pathogens worldwide causing disease in humans and animals mainly by the oral uptake of contaminated food. Consequently, detection methodologies for Salmonella from food items are meaningful for routine laboratories. Here, we describe two different real-time PCR based methods for the detection of Salmonella in food. The procedure begins with a cultural pre-enrichment in buffered peptone water for 18–24 hours at 37 °C followed by a selective enrichment step in Rappaport-Vassiliadis medium for at least six hours at 42 °C. Next, the microbial DNA is extracted and finally Salmonella-DNA is specifically detected by the real-time PCR. Both methods differ in the Salmonella target gene sequence. One assay amplifies a 285-bp-DNA-Fragment of the invA-gene, and the other a 95-bp-DNA-Fragment of the ttrC/ttrA-gene. An internal amplification control indicates the correct carrying out of the PCR. The duration of both detection methods is between 24 and 28 hours.

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Zusammenfassung: Salmonellen z?hlen zu den bedeutendsten bakteriellen Erregern weltweit, die meist durch die Aufnahme von belasteten Lebensmitteln eine Erkrankung bei Menschen und auch Tieren verursachen k?nnen. Verfahren für den Nachweis von Salmonellen in Lebensmitteln haben daher für die diagnostischen Routinelabore eine besondere Bedeutung. Hier beschreiben wir zwei verschiedene real-time PCR basierte Verfahren zum Nachweis von Salmonellen in Lebensmitteln. Das Verfahren beginnt mit einer kulturellen Voranreichung in gepufferten Peptonwasser für 18 bis 24 h bei 37°C und darauf folgender selektiver Anreicherung in Rappaport-Vassiliadis Medium für sechs Stunden bei 42 °C. Anschlie?end erfolgt eine mikrobielle DNA-Extraktion und der Nachweis der Salmonella DNA im Extrakt mittels der realtime PCR. Beide Verfahren unterscheiden sich in der Zielsequenz der nachzuweisenden Salmonella DNA. W?hrend der eine PCR Assay ein 285-bp-DNA-Fragment des invA Gens amplifiziert, weist der zweite Assay ein 95-bp-DNA-Fragment des ttrC/ttrA-Gens von Salmonellen spezifisch nach. Eine interne Amplifikationskontrolle, die in jeder PCR-Reaktion mitgeführt wird, zeigt den funktionsrichtigen Ablauf jeder PCR-Reaktion an. Die Gesamtdauer des Nachweises betr?gt 24 bis 28 Stunden.

Eingegangen: 18. Januar 2007     

Detection of Salmonella in Shellfish Using SYBR Green™ I-Based Real-Time Multiplexed PCR Assay Targeting invA and spvB

A SYBR Green? I-based real-time multiplexed PCR assay was developed targeting invA and spvB for the detection of Salmonella strains in shellfish after both hns and invA genes were identified in all Salmonella strains. Simultaneously, the 16S rRNA gene was used as a PCR internal amplification control (IAC). All 89 Salmonella strains tested in this study exhibited amplification of invA, whereas only 21 (23.6 %) were PCR positive for spvB. The sensitivity of detection of all three targeted genes was 1 ng, which is equivalent to approximately 10⁵ colony-forming unit (CFU) of Salmonella enterica. The analysis showed specific PCR products that were identified by reproducible melt temperature profiles (invA, 84.27?±?1.7 °C; spvB, 88.76?±?1.0 °C; and 16S rRNA gene, 87.16?±?0.8 °C).The sensitivity of detection was 10 pg purified DNA (invA) or 10⁵?CFU in 1 mL pure culture of S. enterica ATCC 14028. The above molecular detection method for Salmonella strains was successfully applied to the oyster homogenates (food matrix). An initial inoculum of 10⁶ and 10²?CFU Salmonella in 1 ml seeded oyster tissue homogenate was detected by multiplexed PCR for all three genes after 5 and 24 h of enrichment, respectively. Natural oysters isolated from Gulf of Mexico during the winter months exhibited negative PCR amplification results suggesting the absence of Salmonella. In contrast to conventional PCR, real-time multiplex PCR assay developed in this study is rapid and sensitive and will help Interstate Shellfish Sanitation Conference undertake appropriate measures to monitor Salmonella in oysters, thereby preventing disease outbreaks and consequently protecting consumer health.     

Development of a novel dual priming oligonucleotide system-based PCR assay for specific detection of Salmonella from food samples

In this study, a novel dual priming oligonucleotide (DPO) system-based polymerase chain reaction (PCR; DPO system-based PCR) assay, which detected the fimY gene of Salmonella, was developed for the fast food testing. The DPO system-based PCR assay allowed a wide range of annealing temperatures at 48–68°C to efficiently amplify fimY gene with an analytical detection limit of 1.2 × 10² CFU/ml for Salmonella in pure cultures and artificially contaminated food matrix. Significantly, the presence of a bubble-like polydeoxyinosine (polyI) linker in the DPO system brought an unparalleled high specificity in the identification of target bacteria, and consequently, the false positives and mismatches of PCR process can be eliminated in priming. Applying the DPO system-based PCR assay to 285 collected food samples revealed that 29 samples were positive in this assay, in accordance with the results of conventional culture-based method, indicating a potential diagnostic capability. The high specificity of the DPO system-based PCR indicates its great potential to be a quick, reliable and practical method for the detection of Salmonella in foods.     

A Pilot Study for Identification of <Emphasis Type="Italic">Salmonella</Emphasis> in Food Processing Plants by Real-Time PCR Screening

Salmonella remains a major public health concern worldwide. Microbiological methods are the gold standard for Salmonella detection. These methods are highly specific, but their sensitivity is variable. Moreover, they are lengthy, labour intensive and not always consistent with the speed of food manufacturing processes. Thus, in the food industry, there is the need for more rapid, sensitive and accurate detection methods. The purpose of this study is to describe a Salmonella-monitoring scheme in different food processing plants based on a screening approach by a commercial real-time polymerase chain reaction (PCR) kit and subsequent confirmation of positive molecular results by the reference microbiological method. This scheme was tested on a total of 4,693 samples, 90 of which were positive with the real-time PCR screening; 52 of the positive samples were eventually confirmed by the microbiological method. The real-time PCR kit was tested in comparison to the microbiological method in order to evaluate its performances and drawbacks. The comparison between cycle threshold (Ct) values of real-time PCR and the microbiological results (Wilcoxon rank sum test) showed a statistically significant difference between the Ct values of bacteriological positive and bacteriological negative samples (p value, <0.05). Furthermore, receiver operating characteristic curve analysis was used to identify the Ct value ensuring the lowest level of misclassification between Salmonella-positive and negative samples. The present study confirms that the real-time PCR kit tested could be used as a screening tool, leading to a rapid and sensitive identification of Salmonella and confining bacteriological confirmation to samples previously identified as positive.     

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.     

Polymerase Chain Reaction for the Rapid Detection and Serovar Identification of Salmonella in Food and Feeding Stuff

The polymerase chain reaction (PCR) is one of the most important rapid methods for the sensitive and specific detection of pathogenic and spoilage microorganisms. The method is increasingly applied in surveillance and monitoring programs to detect pathogens, especially for ensuring the safety and quality of food. The food-borne pathogen Salmonella together with Campylobacter is the most predominant bacterial pathogen in Europe, causing approximately 160,000 confirmed human cases per year. During the last two decades, the importance of Salmonella for food safety triggered the development of dozens of PCR-based detection methods. They promise significant advantages compared to the traditional culture-based methods with respect to speed, easiness, reliability, sensitivity, cost effectiveness, and automation. However, many of them are not applicable because of lacking validation procedures. Meanwhile, PCR has internationally been standardized and guidelines as well as standards for the validation of alternative methods, such as PCR, were established. This review will give an overview of the historical development of PCR-based methods for the detection of Salmonella including validation aspects and summarizes the state-of-the-art for the detection of Salmonella in food and feeding stuff by real-time PCR. Furthermore, current multiplex PCR-based serovar-specific identification methods will be reviewed.     

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.     

Loop-Mediated Isothermal Amplification (LAMP) for the Rapid and Sensitive Detection of Salmonella Typhimurium from Pork

ABSTRACT: Loop-mediated isothermal amplification (LAMP) is a novel molecular detection method that is more rapid and simpler than PCR. Products can be detected by turbidity using one temperature without the need for expensive PCR equipment. Our objective was to sensitively detect Salmonella Typhimurium from pork products within 1 d using the LAMP assay. Pork chop and pork sausage samples (25 g) were inoculated with high (10⁸ to 10⁶ CFU) and low (10⁵ to 10⁰ CFU) inocula of S. Typhimurium. Serial dilutions in phosphate buffered saline were plated on XLT4 agar either immediately or after selective preenrichment in tetrathionate broth (225 mL) for 10-h at 37 °C. Nucleic acid was extracted using the TRIzol^® method from 1-mL samples. The LAMP assay using 6 specific invA gene primers and Bst DNA polymerase reaction mix was carried out at 62 °C for 90 min in a waterbath. Turbid products were detected visually and by agarose gel electrophoresis. Improved Salmonella detection at 10² CFU/25 g for both pork chop and sausage was obtained after 10-h enrichment and 10⁶ CFU/25 g without enrichment for both products. This assay can detect Salmonella from pork within 1 d, significantly faster than traditional methods that take >5 d. This method shows tremendous potential for routine diagnostics and monitoring of Salmonella by the pork industry. Practical Application: The novel loop-mediated isothermal amplification (LAMP) assay is a rapid, specific, and sensitive method that has potential application for routine diagnostics of Salmonella from pork products. The isothermal method does not require expensive equipment such as a PCR thermocylcer but only a simple waterbath for amplification within 90 min. Detection is even simpler by visual eye or turbidimeters that are less expensive than fluorescent spectrophotometers or real-time PCR machines. All these advantages make it a practical approach for routine use by processing industries to rapidly detect Salmonella in their environment and to implement appropriate control strategies. To improve detection sensitivities, preenrichment followed by selective enrichment may be necessary. Even so, the entire assay can be completed at the most within two 8-h working shifts.     

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.     

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.     

Synergistic Reduction of Salmonella in a Model Raw Chicken Media using a Combined Thermal and Acidified Organic Acid Salt Intervention Treatment

ABSTRACT: Salmonella-contaminated poultry products are considered major contributors to foodborne illness. The anti-Salmonella activity of organic acid salts has been studied in food products and poultry feed but rarely in combination with nonchemical treatments. Here, we investigated the combination of acidified organic acid salt solutions with thermal treatment as an effective Salmonella intervention applicable in poultry carcass processing. A model raw chicken media was used to propagate Salmonella prior to the intervention treatment. Salmonella Typhimurium strains LT2 and ATCC nr 14028 grew similarly in the model raw chicken media at 37 and 42 °C, reaching stationary phase 24 h after inoculation. Four log₁₀CFU of either Salmonella Typhimurium strain at stationary phase was exposed to 2.5% organic acid salt solutions (at pH 4) for 1 min at 55 °C. All organic acid salt treatments yielded significant Salmonella Typhimurium reductions, ranging from 1 log (sodium acetate) to almost 4 logs (sodium butyrate). Exposure to pH 4 water at 55 °C or the organic acid salt solutions at room temperature had no effect. The combined thermal and acidified organic acid salt intervention produced a significant, synergistic reduction of Salmonella Typhimurium and may represent an effective method for decontamination of poultry carcasses during processing.     

In-house validation of a real-time PCR method for rapid detection of<Emphasis Type="Italic"> Salmonella</Emphasis> ssp. in food products

A real-time polymerase chain reaction (PCR) method for Salmonella ssp. detection in food samples has been developed and validated in-house. The specificity of the assay was confirmed by tests with 295 different Salmonella strains, including four strains of Salmonella bongori. When tested with extracted Salmonella DNA the lowest detected amount was found to be 5 fg, which is equivalent to approximately one genome copy. The detection limit was further determined by artificial contamination of minced meat with S. Typhimurium cells and of pastry with S. enteritidis using the most probable number approach for cell dose dilutions. It was calculated that even one colony forming unit of Salmonella was still detectable in 25 g food after enrichment culture for 18 h. An additional PCR system for internal positive control, which was included in each reaction and detected in parallel via another reporter fluorescence dye, has no negative impact on the sensitivity of the assay. The method was evaluated with 1,293 naturally contaminated food samples and compared to the conventional cultural method. Of 55 positive PCR samples, 45 were confirmed by the cultural method. The statistical comparison revealed a correlation of 99.2% for specificity, of 100% for sensitivity and of 99.2% for trueness. The results of the comparative analysis and the advantages of the real-time PCR method for detection of Salmonella ssp. under routine laboratory conditions are discussed.     

Rapid Conventional PCR and Real-Time-qPCR Detection of Low Numbers of Salmonella enterica from Ground Beef without Enrichment

Detection of low numbers of Salmonella in complex food matrices such as ground beef by polymerase chain reaction (PCR) without enrichment is particularly difficult because of the presence of PCR inhibitors and fat. This study used soluble starch for the removal of fat in ground beef followed by the use of activated carbon coated with milk proteins for the removal of PCR inhibitors prior to conventional PCR and RealTime qPCR. This methodology without pre-enrichment allowed detection with conventional PCR of 5 CFU/g and 1 CFU/g with the real-time qPCR in ground beef containing 7%, 15%, and 27% fat. The total assay time was 5 h from the seeding of a 25 g sample of ground beef to agarose gel detection of amplicons of a 284 bp invA gene fragment specific for Salmonella and 4.5 h for real-time qPCR detection.     

Development of a rapid FLISA detection of Salmonella spp. based on CdTe/ZnS quantum dots

Salmonella pathogen is old and difficult to control in food safety. The study was aimed to develop a sensitive and rapid CdTe/ZnS quantum dots (QDs) mediated fluorescence-linked immunosorbent assay (FLISA) of Salmonella spp. The optical properties and structure of the CdTe/ZnS QDs were characterized by UV–Vis absorption spectroscopy and transmission electron microscopy. The emission peak wavelength is 575 nm. The CdTe/ZnS QDs-labeled secondary antibody (goat anti-rabbit IgG) was formed by biotin-avidin system. The detection limits of S. Enteritidis and artificial simulated samples are 1.0 × 10³ CFU/ml and 1.78 × 10³ CFU/ml, respectively. The study of FLISA stability shows that the antibody with QDs had very good activity when it was stored in the dark at 4°C for at least 35 days. The FLISA provides a new and easy-operated method for the rapid detection of Salmonella spp. and has broad application in food safety.     

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.     

Validation of Real-Time PCR and Enzyme-Linked Fluorescent Assay-Based Methods for Detection of Salmonella spp. in Chicken Feces Samples

A comparative study of enzyme-linked fluorescent assay (ELFA)-based methods and real-time polymerase chain reaction (PCR)-based methods using three and two different sample preparation protocols, respectively, and the standard culture-based method EN ISO 6579:2002/Amd1:2007, for the detection of Salmonella spp. in chicken feces, was performed on 20 artificially and 68 naturally contaminated chicken feces. Selectivity, relative specificity, relative accuracy, relative sensitivity, and relative detection level were determined. According to criteria established in the methods comparison study included in EN ISO 16140:2003 for validation of alternative microbiological methods, the ELFA-based methods (V1 and V2) as well as a real-time PCR method (PCR2) were comparable to the reference method for the detection of Salmonella in chicken feces. They provided results in 48 h and presented a high sensitivity (97% for all of them). The three methods showed a relative specificity of 94%, V1 being the method which presented the highest relative accuracy (96%). While detection level for V2 and reference method was between 3 and 13 CFU/25 g, PCR2 method was able to detect down to 3 CFU/25 g. In conclusion, both the real-time PCR and the ELFA-based assays can be used as rapid and user-friendly screening methods for detection of Salmonella spp. in chicken feces.     

Rapid detection of food-borne Listeria monocytogenes by real-time quantitative loop-mediated isothermal amplification

The purpose of this study was to develop a real-time quantitative loop-mediated isothermal amplication (LAMP) method for the rapid, sensitive, and convenient detection of Listeria monocytogenes in food. The LAMP method could amplify the hlyA gene of L. monocytogenes successfully at 63°C with a loopamp real-time turbidimeter. The detection limits of the LAMP for hlyA gene were 6 colony forming units (CFU)/tube. A standard curve was generated for L. monocytogenes LAMP by plotting the graph based different log CFU values of L. monocytogenes and time of positivity through real-time monitoring of the amplication. Then, the LAMP method was employed to test 94 retail food samples effectively. Sensitivity in detection of L. monocytogenes by the LAMP was higher than that of PCR and none of the conventional methodpositive samples was missed by the LAMP method.     

Modelling the Growth of Salmonella spp. and Escherichia Coli O157 on Lettuce

This study aimed to model the growth of Salmonella and Escherichia coli O157 on lettuce at different temperatures. Microorganisms were inoculated separately on lettuce and stored at 5, 10, 25, and 37°C. Growth curves were built by fitting the data to the Baranyi's DMFit model and Ratkowsky equation was used as secondary model. The models were able to assess the growth of both microorganisms and data showed that bacteria did not growth for 24hours at 10°C, what can be a suitable temperature for lettuce distribution on food services. However, prolonged periods demonstrated growth at every temperatures examined.     

Comparative Study of a DNA Hybridization Method and the Conventional Culture Procedure for Detection of Salmonella in Foods

A commercial DNA hybridization assay (DNAH) for rapid detection of Salmonella in foods was compared to the conventional culture procedure. The DNAH method employed preenrichment, selective, and post-enrichment steps (44°hr) prior to performing the assay (4hr). Confirmation of positive DNAH assays was accomplished using standard culture methods for isolation of Salmonella. More than 1,600 samples were tested, representing 23 food types and including naturally contaminated, artificially inoculated, and uninoculated foods. Based on the data generated, the DNAH method was as productive as the standard culture method for detection of Salmonella in all foods and was significantly better than the culture method for certain foods.