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.     

Application of quantitative real-time PCR in the detection of prion-protein gene species-specific DNA sequences in animal meals and feedstuffs

This study describes a method for quantitative and species-specific detection of animal DNA from different species (cattle, sheep, goat, swine, and chicken) in animal feed and feed ingredients, including fish meals. A quantitative real-time PCR approach was carried out to characterize species-specific sequences based on the amplification of prion-protein sequence. Prion-protein species-specific primers and TaqMan probes were designed, and amplification protocols were optimized in order to discriminate the different species with short PCR amplicons. The real-time quantitative PCR approach was also compared to conventional species-specific PCR assays. The real-time quantitative assay allowed the detection of 10 pg of ruminant, swine, and poultry DNA extracted from meat samples processed at 130 degrees C for 40 min, 200 kPa. The origin of analyzed animal meals was characterized by the quantitative estimation of ruminant, swine, and poultry DNA. The TaqMan assay was used to quantify ruminant DNA in feedstuffs with 0.1% of meat and bone meal. In conclusion, the proposed molecular approach allowed the detection of species-specific DNA in animal meals and feedstuffs.     

Simultaneous detection of mastitis pathogens, Staphylococcus aureus, Streptococcus uberis, and Streptococcus agalactiae by multiplex real-time polymerase chain reaction

The objective of this study was to develop a multiplex real-time polymerase chain reaction (PCR) method for simultaneous detection of Staphylococcus aureus, Streptococcus agalactiae, and Streptococcus uberis directly from milk. A genetic marker specific for Staph. aureus was used for primers and dual-labeled probe design. The target for Strep. agalactiae primers and dual-labeled probe was selected from the cfb gene encoding the Christie-Atkins-Munch-Petersen factor. The plasminogen activator gene was the target for primers and dual-labeled probe design for Strep. uberis. Quarter milk samples (n = 192) were analyzed by the multiplex real-time PCR assay and conventional microbiological methods. An additional 57 quarter milk samples were analyzed in a separate real-time PCR assay for Strep. agalactiae only. Using an overnight enrichment step, the real-time PCR technique correctly identified 96.4% of all quarter milk samples; 91.7% of Staph. aureus, 98.2% of Strep. agalactiae, and 100% of Strep. uberis. Results of conventional microbiological methods were used to determine the sensitivity and specificity of the multiplex real-time PCR procedure. The sensitivity of the procedure to correctly identify Staph. aureus, Strep. agalactiae, and Strep. uberis directly from milk was 95.5%, and the specificity was 99.6%. Results of this study indicate that the multiplex real-time PCR procedure has the potential to be a valuable diagnostic technique for simultaneous identification of Staph. aureus, Strep. agalactiae, and Strep. uberis directly from quarter milk samples.     

Rapid detection of Listeria monocytogenes in food using culture enrichment combined with real-time PCR

A rapid method for the detection of Listeria monocytogenes in foods combining culture enrichment and real-time PCR was compared to the ISO 11290-1 standard method. The culture enrichment component of the rapid method is based on the ISO standard and includes 24 h incubation in half-Fraser broth, 4 h incubation in Fraser broth followed by DNA extraction and real-time PCR detection of the ssrA gene of L. monocytogenes. An internal amplification control, which is co-amplified with the same primers as the L. monocytogenes DNA, was also included in the assay. The method has a limit of detection of 1–5 CFU/25 g food sample and can be performed in 2 working days compared to up to 7 days for the ISO standard. A variety of food samples from retail outlets and food processing plants (n = 175) and controls (n = 31) were tested using rapid and conventional methods. The rapid method was 99.44% specific, 96.15% sensitive and 99.03% accurate when compared to the standard method. This method has the potential to be used as an alternative to the standard method for food quality assurance providing rapid detection of L. monocytogenes in food.     

Identification of chicken,duck, pigeon and pig meat by species-specific markers of mitochondrial origin

In the present study, PCR based method for meat species identification of chicken, duck, pigeon and pig was achieved by developing species-specific markers. Using mitochondrial sequences species-specific primers were designed and the sizes of them were 256 bp, 292 bp, 401 bp and 835 bp for chicken, duck, pigeon and pig, respectively. The species-specific PCR products were sequenced to confirm the specificity of the product amplified. These markers were subsequently tested for cross amplification by checking them with beef, mutton, chevon, pork, rabbit, chicken, duck, turkey and pigeon meat. DNA markers developed in this study can help identify the species of fresh, cooked and autoclaved meat of chicken, duck and pigeon and fresh and cooked meat of pig. The process of identification is simple, economical and quick as compared to other methods such as RAPD, PCR-RFLP and sequencing method of species identification.     

A novel real-time polymerase chain reaction-based method for the detection and quantification of lactose-fermenting Enterobacteriaceae in the dairy and other food industries

The presence of lactose-fermenting Enterobacteriaceae and coliforms is routinely assessed to determine the hygienic quality of water and foods, particularly dairy products. This paper reports the use of lacZ-specific primers in an SYBR green I-based real-time PCR method for the easy and rapid detection of coliforms in dairy products. A large number of bacterial species were assayed to establish the specificity of the method. The sensitivity of the method was assessed using artificially contaminated cheeses. The limit of detection was 1 coliform cell in cheese samples enriched for 8 h in a culture medium. The entire procedure, including sample processing, enrichment, DNA extraction, and real-time PCR amplification, can be completed within 10 to 12 h, making it a single-day assay.     

Detection of meat species using TaqMan real-time PCR assays

Species-specific real-time PCR (TaqMan) assays were developed for detection of beef, pork, lamb, chicken and turkey. Assays were developed around small (amplicons <150 base pairs) regions of the mitochondrial cytochrome b (cytb) gene. Speciation was achieved using species-specific primers. For detection purposes, two TaqMan probes were developed; the first was specific to the mammalian species (beef, lamb and pork), the second to the poultry species (chicken and turkey). Normal end-point TaqMan PCR conditions were applied; however, PCR was limited to 30 cycles. Applying the assays to DNA extracts from raw meat admixtures, it was possible to detect each species when spiked in any other species at a 0.5% level. The absolute level of detection, for each species, was not determined; however, experimentally determined limits for beef, lamb and turkey were below 0.1%.     

Real-time polymerase chain reaction for the quantitative detection of tetA and tetB bacterial tetracycline resistance genes in food

A new, rapid, sensitive and specific method was developed to directly detect and quantify tetA and tetB in food. Both tet genes are two of the most frequently present tetracycline resistance genes in Gram-negative bacteria. A set of primers and Taqman probes was designed for each gene. The standard curves were performed using Escherichia coli BM13 (C600 RifR)/RP4 and E. coli NCTC 50365, which carry tetA and tetB, respectively. Meat and fish samples inoculated with these reference strains were used as a matrix to construct the standard curves for the analysis of 20 samples of chicken meat and 10 samples of hake (Merlucius merlucius). The limits of detection in pure culture were 5 cfu/mL (0.7 log cfu/mL) in the case of tetA, 50 cfu/mL (1.7 log cfu/mL) for tetB and 5 × 10² cfu/g (2.7 log cfu/g) for both genes in food samples. The results obtained by real-time quantitative polymerase chain reaction (qPCR) were compared to counts of tetracycline-resistant bacteria obtained by plating extracts of poultry and hake samples in culture media supplemented with 16 mg/L of tetracycline. Counts of tetracycline-resistant bacteria obtained by qPCR showed a positive correlation, especially interesting when compared with microbiological counts of tetracycline-resistant Enterobacteriaceae in poultry meat (r = 0.5509) and with tetracycline-resistant mesophilic aerobic bacteria in hake samples (r = 0.7146). The obtained results demonstrate that this method could be a useful tool for the direct quantification of the amount of bacterial strains that carry tetA and/or tetB genes in food samples.     

Detection of roe deer,red deer,and hare meat in raw materials and processed products available in Poland

To allow detection of meat from the most popular game species in Poland, we developed a PCR-based method for identification of roe deer (Capreolus capreolus), red deer (Cervus elaphus), and hare (Lepus europaeus). The designed primers were based on the noncoding, mitochondrial D-loop region. Amplicon sizes ranged from 116 to 255 bp. The primers exhibited no cross-reactivity with the DNA from common slaughter and other game species. The detection limit of the assay was established to be below 0.001 % in raw red deer (C. elaphus) and hare (L. europaeus) meat, and below 0.01 % in raw roe deer (C. capreolus) meat, whereas <0.5 % of hare and red deer meat in processed samples could be detected. The PCR-based assay was used for authentication of 17 samples of raw game meat and 32 samples of game meat-containing products available in Polish markets. Analysis of all tested raw meat and processed products revealed the presence of DNA of investigated species in concordance with producers’ declarations.     

Authentication of carabeef (water buffalo, <Emphasis Type="Italic">Bubalus bubalis</Emphasis>) using highly specific polymerase chain reaction

In order to ensure consumer satisfaction and fraud detection, correct identification of meat animal species becomes significant. Buffalo being one of the major meat animal species in Asia, a species-specific polymerase chain reaction (PCR) was developed for the accurate identification of carabeef (water buffalo, Bubalus bubalis) targeting mitochondrial D-loop region. Unique diagnostic PCR developed in this study employed novel primers to yield a 534-bp buffalo-specific PCR product, and chances of cross-amplification were excluded by including as many as 25 animal species. Applicability of PCR was established in raw, cooked (60, 80 and 100 °C for 30 min), autoclaved (121 °C for 30 min) and microoven-processed meats with a sensitivity of detection of 0.1% adulteration (10 pg bubaline DNA). Keeping in view adulteration, socio-economic, religious, quality assurance, forensic and legal issues, the novel buffalo-specific PCR developed in this study was found highly promising in authenticating buffalo meat, ensuring consumer satisfaction and labeling process.     

Quantitative detection of poultry meat adulteration with pork by a duplex PCR assay

A species-specific duplex polymerase chain reaction (PCR) assay was developed for the simultaneous detection of pork and poultry meat species using the mitochondrial cytb and 12S rRNA as target genes for pork and poultry, respectively. By the amplification of binary reference meat mixtures, a linear normalised calibration curve was obtained using the fluorescence intensities of PCR products for pork (149 bp) and poultry (183 bp) species. The proposed method allowed the quantification of pork meat addition to poultry meat in the range of 1–75%, with a sensitivity of 0.1%. The in-house validation using samples with known amounts of pork meat (1.0%, 2.5%, 7.5%, 20.0% and 40%) evidenced a high reproducibility of the methodology (coefficient of variation from 4.1% to 7.6%). The successful application of the duplex PCR was also demonstrated by the high correlation (R² = 0.99) obtained from regression analysis between the predicted and the actual values of pork meat addition in blind meat mixtures. The suggested methodology presents a low cost, fast, easy and reliable alternative to estimate the level of poultry meat adulteration by the addition of pork meat.     

A filtration-based real-time PCR method for the quantitative detection of viable Salmonella enterica and Listeria monocytogenes in food samples

We developed a novel filtration-based method that can eliminate dead or severally damaged Salmonella enterica and Listeria monocytogenes in food samples. This new method can recover all viable bacteria in less than 30 min, and can be coupled with a subsequent bacterial DNA extraction and real-time PCR. No statically significant differences (p < 0.01) were found between real-time PCR results obtained separately from S. enterica and L. monocytogenes when different ratios of living and dead cells were used. The analytical sensitivity in both cases was 1 genome equivalent (GE), and the quantification was linear (R² > 0.9969) over a 5-log dynamic range with PCR efficiencies >0.9754. When compared with the standard microbiological methods for the detection of these foodborne pathogens, the relative accuracy was excellent ranging from 95.72% to 104.48%. Finally, we applied the pre-treatment method to the direct detection of viable forms of these foodborne pathogens in food samples using yogurt as a model, the results being similar to those obtained using pure cultures.     

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.     

PorA specific primers for the identification of Campylobacter species in food and clinical samples

Campylobacteriosis is a public health problem with considerable socio-economic impact. As the European Food Safety Authority has emphasized the importance of a surveillance programme for campylobacteriosis, the aim of the present study was the optimization of a specific and sensitive PCR protocol able to detect Campylobacter species responsible for gastrointestinal infections. Raw poultry meat samples were analysed for the presence of Campylobacter sp., by plating onto mCCD (Modified Charcoal-Cefoperazone-Deoxycholate) Agar and Campylobacter Selective Preston Agar and using four sets of species-specific primers for Campylobacter jejuni, Campylobacter coli, Campylobacter upsaliensis and Campylobacter lari designed to bridge the porA gene. The resulting primers demonstrated a sensitivity of 0.01 ng/μl for the C. coli-specific, C. lari-specific, and C. upsaliensis-specific primer sets and 0.5 ng/μl for the C. jejuni-specific primer sets using DNA from pure cultures. Non-specific amplification of non-target DNA was not observed indicating excellent specificity. The primers were useful for the analyses of poultry meat samples both for direct plating onto mCCDA, and for DNA extracted directly from the cells grown for 48 h in Preston enrichment broth. The sets of primers were also useful when used for species identification of human isolates.     

Development of multiplex loop-mediated isothermal amplification-RFLP (mLAMP-RFLP) to detect Salmonella spp. and Shigella spp. in milk

A multiplex loop-mediated isothermal amplification-RFLP (mLAMP-RFLP) was developed and validated for simultaneous detection of Salmonella strains and Shigella strains in milk. In this system, two sets of LAMP primers were designed to specifically target invA of Salmonella spp. and ipaH of Shigella spp. Under isothermal conditions at 63 °C, ladder pattern of DNA bands could be amplified within 60 min in the presence of genomic DNAs of Salmonella strains and Shigella strains, which could be distinguished between Salmonella spp. and Shigella spp. simultaneously based on the different ladder pattern of DNA bands and subsequent restriction enzyme analysis. The overall analysis time was approximately 20 h including the enrichment of the bacterial cells, which greatly saved detection time. The sensitivity of mLAMP was found to be 100 fg DNA/tube with genomic DNAs of Salmonella strains and Shigella strains, comparatively, multiplex PCR was 1 pg DNA/tube. The mLAMP allowed the detection of milk sample artificially contaminated by Salmonella strains and Shigella strains at initial inoculation levels of approximate 5 CFU/10 mL. In conclusion, the mLAMP described here can potentially facilitate simultaneous monitoring of Salmonella and Shigella in a large number of food samples, which could be used as a primary screening method and as a supplement to classical detection method.     

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.     

A fast and accurate method for controlling the correct labeling of products containing buffalo meat using High Resolution Melting (HRM) analysis

The substitution of high priced meat with low cost ones and the fraudulent labeling of meat products make the identification and traceability of meat species and their processed products in the food chain important. A polymerase chain reaction followed by a High Resolution Melting (HRM) analysis was developed for species specific detection of buffalo; it was applied in six commercial meat products. A pair of specific 12S and universal 18S rRNA primers were employed and yielded DNA fragments of 220 bp and 77 bp, respectively. All tested products were found to contain buffalo meat and presented melting curves with at least two visible inflection points derived from the amplicons of the 12S specific and 18S universal primers. The presence of buffalo meat in meat products and the adulteration of buffalo products with unknown species were established down to a level of 0.1%. HRM was proven to be a fast and accurate technique for authentication testing of meat products.     

Ohmic cooking of whole beef muscle — Evaluation of the impact of a novel rapid ohmic cooking method on product quality

Cylindrical cores of beef semitendinosus (500 g) were cooked in a combined ohmic/convection heating system to low (72 °C, LTLT) and high (95 °C, HTST) target end-point temperatures. A control was also cooked to an end-point temperature of 72 °C at the coldest point. Microbial challenge studies on a model meat matrix confirmed product safety. Hunter L-values showed that ohmically heated meat had significantly (p < 0.05) lighter surface-colours (63.05 (LTLT) and 62.26 (HTST)) relative to the control (56.85). No significant texture differences (p ≥ 0.05) were suggested by Warner–Bratzler peak load values (34.09, 36.37 vs. 35.19 N). Cook loss was significantly (p < 0.05) lower for LTLT samples (29.3%) compared to the other meats (36.3 and 33.8%). Sensory studies largely confirmed these observations. Cook values were lower for LTLT (3.05) while HTST and the control were more comparable (6.09 and 7.71, respectively). These results demonstrate considerable potential for this application of ohmic heating for whole meats.     

Puffer fish-based commercial fraud identification in a segment of cytochrome b region by PCR–RFLP analysis

To identify the mislabeled or fraudulently substituted toxic puffer fish in thermally processed fish products, a polymerase chain reaction (PCR) method using restriction sites and sequence analysis has been developed in this study. A 376-bp fragment of the cytochrome b gene was produced after PCR amplification. Fish tissue samples were prepared under autoclaving conditions at 121 °C for 10–90 min at 10 min intervals. DNA fragments could not be detected after 90 min of autoclaving at 121 °C. For PCR product digestion, BsaJ I, Aci I, Hinf I, Taq I, and Sap I endonucleases were used to yield species-specific profiles for the identification of puffer fish species from 60 commercial market samples. Results from this study showed that the restriction fragment length polymorphism technique can be used to identify 17 puffer fish species from commercial products even after severe thermal processing.