Relative Accuracy,Specificity and Sensitivity of a 5′ Nuclease Real-Time PCR Assay for Listeria monocytogenes Detection in Naturally Contaminated Pork Cuts

In the present study, the relative sensitivity, specificity and accuracy of a real-time PCR assay for Listeria monocytogenes detection in naturally contaminated pork cuts were evaluated in comparison to the ISO 11290-1:2004 reference culture method. One hundred and sixty meat samples were collected from ten different lots over a year. The PCR method included a 24-h primary enrichment step, a DNA extraction step, and a final 5′ nuclease real-time PCR assay, including an internal amplification control (IAC) and targeting the hlyA gene. Based on the analysis of 480 sub-units (three sub-units for each sample), the relative sensitivity, specificity and accuracy of the real-time PCR assay were 77.3 %, 67.0 % and 73.1 %, respectively, corresponding to a Cohen’s kappa value of 0.69 (good agreement). Considering that real samples from the worse storage scenarios were included, these results suggest the PCR method as a rapid and accurate alternative method for the quick check of L. monocytogenes in meat lots before distribution.     

Validation of a Loop-Mediated Amplification/ISO 6579-Based Method for Analysing Soya Meal for the Presence of <Emphasis Type="Italic">Salmonella enterica</Emphasis>

An alternative method for detection of Salmonella spp. in animal feed, based on the use of loop-mediated amplification (LAMP) in conjunction with a standard culturing procedure, was compared with the standard ISO 6579 as reference method, using soya meal as the test matrix. In the method comparison study, the sensitivities for both the alternative and reference methods were 100 %. The relative level of detection was 1.000. Tested against 100 Salmonella and 30 non-Salmonella strains, the LAMP-based method was 99 % inclusive and 100 % exclusive. The interlaboratory trial involved ten laboratories from eight European countries, testing eight samples at three contamination levels: 0 cfu/100 g, 1–5 cfu/100 g and 14–68 cfu/100 g. The trial specificity, or percentage correct identification of uncontaminated samples, was 96.3 % for both the reference methods and the LAMP/ISO 6579 alternative method, thus demonstrating its suitability for adoption as a procedure for rapid identification of Salmonella uncontaminated soya meal.     

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.     

Salmonella spp. contamination in fresh pork and chicken sausages marketed in Niterói and Rio de Janeiro,Brazil

Fresh sausages are one of the most popular meat products consumed in Brazil. However, the use of contaminated raw material, unhygienic handling during processing, and inadequate storage could be hazardous to consumer’s health, since fresh-sausage processing does not include heat treatment and the product may carry several human pathogens such as Salmonella spp.. In this study, a total of 80 fresh sausages prepared with pork and chicken meat were evaluated regarding Salmonella spp. presence by PCR after selective enrichment and bacteriological culture. Twenty-one sausage samples (27 %) proved to be contaminated with Salmonella spp.. Sixteen (76 %) contaminated samples were detected by PCR combined with enrichment broth culturing, whereas conventional microbiological screening detected only 8 (38 %). In this study, evidence was raised regarding packing conditions of fresh sausages, since it seems that there is no significant difference in Salmonella spp. contamination among samples in their original packing (23 %), wrapped in plastic or unpacked (27 %). Similarly, meat composition seems not to have a determinant influence in contamination, since 20 % of the chicken and 29 % of the pork sausages were contaminated. The contamination rate of Tuscan-type sausages was 37 %, while contamination was found in 21 % of ham sausages, suggesting that the cut of pork did not have a determinant influence in the contamination by Salmonella in fresh sausages. The results shown here indicate that fresh sausages marketed in Niterói and Rio de Janeiro may be contaminated with Salmonella spp., requiring more attention by sanitary authorities in order to assure the safety of this food.     

Development and evaluation of DNA and RNA real-time assays for food analysis using the hilA gene of Salmonella enterica subspecies enterica

The objective of this study was the development of DNA and RNA real-time PCR methods for detection of food-borne Salmonella sp. as rapid alternatives to the traditional cultural method (ISO 6579, 2004) in fresh meat carcasses and processed meat samples. These PCR methods were based on the hilA sequence, with primers and hybridisation probes designed against this gene target. The primers and probes were evaluated for their efficiency and dynamic range and subsequently the specificity of the assay was tested using 106 Salmonella enterica subspecies enterica strains and 30 non-salmonellae strains. An internal amplification control (IAC) was also developed for incorporation. The optimum copy number of IAC was determined to be 500 copies per reaction. A complementary enrichment protocol was adapted from the existing standard ISO 6579:2004 and consisted of enrichment in Buffered Peptone Water (BPW) 22 ± 2 h and a second selective enrichment for 6 h in Rappaport Vassiliadis with Soya (RVS). The DNA and RNA-based real-time PCR protocols, were applied to meat samples inoculated with Salmonella enterica subspecies enterica strains, including swabs from meat carcasses and minced beef samples which were heat treated or frozen. The developed methods have the potential as useful alternatives to the standard ISO 6579:2004 method for the detection of Salmonella enterica subspecies enterica on carcass swabs and raw meat using hilA as a target. The DNA assay is a useful tool for the screening of meat samples in the abattoir within 3 days of slaughter or in a food production process and the RNA-based assay has the potential to detect viable Salmonella enterica subspecies enterica in ready-to-eat products.     

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.     

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.     

Rapid Sample Preparation for Molecular Biological Food Analysis Based on Magnesium Chloride

The prerequisites of rapid and reliable direct quantification of foodborne pathogens deem prior sample preparation a necessity. This study describes a new system for the modular and rapid (3 h) sample preparation method “Matrix-Lysis” for the quantification of Listeria monocytogenes and Salmonella typhimurium from food. In this study a new chemical concept is introduced for Matrix-Lysis, based on solubilization of proteins through the preferential interaction with MgCl₂. The results of this study show that the new system has many advantages compared to the previously described Matrix-Lysis systems, both regarding the performance of the whole method as well as the chemistry employed for solubilization of the foodstuffs. Artificial contamination experiments showed average recovery rates of 82 % for S. typhimurium and 73 % for L. monocytogenes from several foodstuffs with detection limits <10 CFU/g. Examination of naturally contaminated acid curd cheese samples, from a recent L. monocytogenes outbreak in Austria and Germany, with the new buffer system resulted in 100 % relative accuracy, 100 % relative specificity, and 100 % relative sensitivity compared to the ISO 11290-1 standard method. The results demonstrate the excellent applicability of Matrix-Lysis as sample preparation for the direct quantification of foodborne pathogens with molecular biological, as well as microbiological methods and the new chemical concept described enables the widespread use of this method for routine applications.     

Nonaplex real-time PCR detection of Listeria monocytogenes, Campylobacter, Salmonella and enteropathogene E. coli after universal enrichment in food samples

A universal cultivation media for Listeria monocytogenes, Salmonella and enteropathogenic Escherichia coli in conjunction with a highly efficient nonaplex PCR for the determination of L. monocytogenes, Campylobacter, Salmonella and enteropathogenic E. coli was developed and compared to classical microbiological assays. The achieved detection limit was at 2 colony-forming units per g for all analytes. The method allows screening of food samples within 24 h and is partly automatable. A ring trial showed that it can be easily established in other laboratories showing its robustness. The presented method yields results similar to the classical ISO methods based on cultivation.     

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.     

Detection and differentiation of Salmonella serotypes using surface enhanced Raman scattering (SERS) technique

This research was conducted to prove that developed silver biopolymer nanoparticle substrate for surface enhanced Raman scattering (SERS) technique could detect and differentiate three different serotypes of Salmonella. Nanoparticle was prepared by adding 100 mg of silver nitrate to a 2 % polyvinyl alcohol solution, then adding 1 % trisodium citrate to reduce silver nitrate and produce silver encapsulated biopolymer nanoparticles. Then, nanoparticle was deposited on a stainless steel plate and used as SERS substrate. Fresh cultures of Salmonella typhimurium, Salmonella enteritidis and Salmonella infantis were washed and suspended in 10 mL of sterile deionized water. Approximately 5 μl of the bacterial suspensions were placed on the substrate individually and exposed to 785 nm laser excitation. SERS spectral data were recorded between 400 and 1,800 cm^?1. SERS signals were collected from 15 different spots on the substrate for each sample. PCA model was developed to classify Salmonella serotypes. PC1 identified 92 % of the variation between the Salmonella serotypes, and PC2 identified 6 % and in total 98 % between the serotypes. Soft independent modeling of class analogies of validation set gave an average correct classification of 92 %. Comparison of the SERS spectra of Salmonella serotypes indicated that both isolates have similar cell walls and cell membrane structures which were identified by spectral regions between 520 and 1,050 cm^?1. However, major differences were detected in cellular genetic material and proteins between 1,200 and 1,700 cm^?1. SERS with silver biopolymer nanoparticle substrate could be a promising tool in pathogen detection and it would potentially be used to classify them.     

A new 7-plex PCR assay for simultaneous detection of shiga toxin-producing Escherichia coli O157 and Salmonella Enteritidis in meat products

A 7-plex PCR assay was developed to achieve an effective detection and identification of serotype Enteritidis of Salmonella spp. and shiga toxin-producing type of Escherichia coli O157 in meat products. Six DNA sequences in the invA and sdfI genes of Salmonella Enteritidis as well as rfbE, eae, stx1, and stx2 genes of E. coli O157:H7 were employed to design primers. The multiplex PCR assay could specifically and sensitively detect and identify target pathogens. Applying the assay to meat samples, the multiplex PCR assay was able to simultaneously detect and identify the two pathogens at a sensitivity of three CFU/10?g raw meats after simple 16?h enrichment in buffered peptone water. Further applying in 21 retail meat samples revealed that two samples were positive for non-shiga toxin producing E. coli O157, one sample was positive for Stx2 producing E. coli O157 and five samples were positive for Salmonella enterica Enteritidis. Taken together, the 7-plex PCR assay is a rapid and reliable method for effectively screening single or multiple pathogens occurrences in various meat products, and could also identify the Salmonella enterica Enteritidis from all Salmonella spp. and shiga toxin producing type from all E. coli strains. Considering as a non expensive screening tool, the multiplex PCR assay has a great potential in complement for food stuff analysis by conventional microbiological tests.     

A Real-Time PCR/SYBR Green I Method for the Rapid Quantification of Salmonella enterica in Poultry Meat

It has been developed a method for quantitative detection of Salmonella enterica in poultry meat based on real-time PCR (qtPCR) with species-specific primers and SYBR® GreenER? chemistry. Two methods for bacterial DNA extraction were compared: one based on a commercial kit (AccuPrep®) and the other on silica–magnetite nanoparticles. Primers were designed on sequence of invA gene encoding for an inner membrane protein associated with invasiveness of Salmonella. Serial dilutions of DNA from pure cultures of Salmonella and from broiler breast samples spiked with serial dilutions of Salmonella were analyzed in different replicates and with different PCR equipments. Robustness of the method was evaluated and compared in terms of repeatability, reproducibility, and consistency with conventional plate count methods and for applicability to the different equipments. The matrix effect upon each reaction specificity was assessed with addition of DNA from a noncompetitive internal amplification control. The limit of detection (LOD) was determined between 10 and 40 colony-forming units (CFUs)/ml; whereas, the limit of quantification (LOQ) was 10² CFUs/ml. Quantification with qtPCR was in the same order of magnitude as enumeration with plate counting but with an overestimation.     

Multicenter validation study of two blockcycler- and one capillary-based real-time PCR methods for the detection of Salmonella in milk powder

A collaborative study including 13 German laboratories was conducted to evaluate the performance of two non-patented real-time PCR methods for the detection of Salmonella in milk powder targeting the ttrC/ttrA- or the invA gene. The enrichment procedure and sample DNA preparation method prior to the real-time PCR was the same for both systems and the identical DNA extraction samples were analysed. The traditional cultural method according to EN ISO 6579:2002 for the detection of Salmonella in food was performed in each laboratory as the reference. The participants received twelve coded milk powder samples each of 25 g for the analysis. Four of them were Salmonella negative (level L0), four artificially contaminated with <3 MPN/g Salmonella Typhimurium (level L1) and four artificially contaminated with 3.6 MPN/g S. Typhimurium (level L2) to the beginning of the experiment. Of the 13 laboratories 12 used various models of real-time PCR blockcyclers conducting both real-time PCR assays and three laboratories the Light Cycler 2.0 system (Roche Bioscience) conducting the ttr-based real-time PCR assay only. The relative accuracy for both real-time PCR assays performed on blockcyclers was for level L0 97.5%. For level L1 the relative accuracy was 94.1% and for level L2 it was 100% for both assays. The relative accuracy on the Light Cycler 2.0 system was 100% for all levels applied to the ttr-real-time PCR.     

Antimicrobial Polylactic Acid Packaging Films against Listeria and Salmonella in Culture Medium and on Ready-to-Eat Meat

The contamination of Listeria monocytogenes and Salmonella spp. in ready-to-eat (RTE) meat products has been a concern for the meat industry. In this study, edible chitosan-acid solutions incorporating lauric arginate ester (LAE), sodium lactate (NaL), and sorbic acid (SA) alone or in combinations were developed and coated on polylactic acid (PLA) packaging films. Antimicrobial effects of coated PLA films on the growth of Listeria innocua, L. monocytogenes, and Salmonella Typhimurium in a culture medium (tryptic soy broth, TSB) and on the surface of meat samples were investigated. Antimicrobial PLA films containing 1.94 mg/cm² of chitosan and 1.94 μg/cm² of LAE were the most effective against both Listeria and Salmonella in TSB and reduced them to undetectable level (<0.69 log CFU/ml). The same PLA films with LAE significantly (p?L. innocua, L. monocytogenes, and S. Typhimurium on RTE meat during 3 and 5 weeks’ storage at 10 °C, achieving 2–3 log reduction of Listeria and 1–1.5 log reduction of Salmonella as compared with controls. PLA films coated with 1.94 mg/cm² of chitosan, 0.78 mg/cm² of NaL, and 0.12 mg/cm² of SA significantly reduced the growth of L. innocua but were less effective against Salmonella. The combination of NaL (0.78 mg/cm²) and SA (0.12 mg/cm²) with LAE (1.94 μg/cm²) did not generate additional or synergetic antimicrobial effect against Listeria or Salmonella on the meat surface. L. innocua had a similar sensitivity to the film treatments as L. monocytogenes, suggesting that L. innocua may be used as a surrogate of L. monocytogenes for further scaleup and validation studies. The film treatments were more effective against the microorganisms in TSB culture medium than in RTE meat, which suggests that in vivo studies are a necessary step to develop antimicrobial packaging for applications in foods.     

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.     

Application of the SureTect Detection Methods for <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> and <Emphasis Type="Italic">Listeria</Emphasis> spp. in Meat,Dairy, Fish,and Vegetable Products

The aim of this study was to evaluate the application of the Listeria monocytogenes and Listeria spp. SureTect detection methods for a rapid and sensitive detection in an ample range of food products: raw pork and poultry meat, Spanish chorizo, pate, smoked salmon, raw sheep milk cured cheese, and ready-to-eat lettuce salad. The combination of a 24-h enrichment in the 24 Listeria Enrichment Broth (LEB) coupled to a rapid bacterial DNA extraction and real-time polymerase chain reaction (RTi-PCR) using the specific SureTect methods detected down to 2–6 L. monocytogenes CFU per sample in less than 27 h on the food categories tested. Furthermore, the applicability of L. monocytogenes and Listeria spp. SureTect detection methods in real samples was assessed using 303 food samples, obtaining at least the same analytical performance as the international reference method ISO 11290-1.     

A Robust and Poisson Validated Quantitative 5′ Nuclease TaqMan® Real-Time PCR Assay Targeting fimA for the Rapid Detection of Salmonella spp. in Food

A newly designed TaqMan® probe and an internal amplification control were implemented in a conventional PCR system targeting the major fimbrial subunit encoding gene fimA. This assay has an inclusivity and exclusivity of 100 % (n?=?126). The limit of detection (LOD) and the absolute quantification limit, both determined by advanced Poisson analyses, were three bacterial cell equivalents per quantitative real-time PCR reaction. The fimA assay achieved 100 % accuracy and performed very robustly, producing reproducible, reliable data for quantification of Salmonella spp., even at the LOD.     

Simultaneous Detection of Salmonella,Listeria monocytogenes,and Staphylococcus aureus by Multiplex Real-Time PCR Assays Using High-Resolution Melting

A method combining multiplex real-time polymerase chain reaction (PCR) with high-resolution melting (HRM) analysis for rapid and specific simultaneous detection of Salmonella, Listeria monocytogenes, and Staphylococcus aureus was developed. The method included a melting-curve analysis of products and was evaluated by specificity, sensitivity and reproducibility analyses. Sensitivity and reproducibility analyses was both conducted by genomic DNA extracted from serial dilutions for each target pathogen. Assays with artificially inoculated and naturally contaminated samples after enrichment were also conducted. In the specificity test, there was no nonspecific amplification of the 44 nontarget pathogens, whereas the actual T _m values were 79.38?±?0.14, 82.54?±?0.15, and 77.36?±?0.14 °C for Salmonella, L. monocytogenes, and S. aureus, respectively. The sensitivity of the method was 3.5?×?10² CFU ml^?1 for Salmonella and L. monocytogenes and 3.5?×?10³ CFU ml^?1 for S. aureus. The coefficients of variation of T _m values ranged 0.51–1.03 % for Salmonella, 1.63–2.11 % for L. monocytogenes, and 0.75–2.17 % for S. aureus in intraassay, and ranged 0.81–2.43 % for Salmonella, 1.97–2.35 % for L. monocytogenes, and 0.93–3.93 % for S. aureus in interassay. The detection limit in artificially inoculated samples (n?=?50) was 5 CFU (25 g)^?1 food for the three tested pathogens. In the naturally contaminated samples (n?=?120),Salmonella DNA was detected by HRM, sequencing, and conventional culture-based methods at a positive rate of 25.00, 25.00, and 24.17 %, respectively; the corresponding rates for L. monocytogenes were 14.17, 14.17, and 14.17 %, respectively, while those for S. aureus were 16.7, 16.7, and 16.7 %, respectively.