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The developed polymerase spiral reaction-based technique specifically amplified the ceuE gene of C. coli and involved a three-step centrifugation method for DNA extraction. PSR, real-time and end-point PCR were able to detect 62 fg, 620 fg and 6.2 pg C. coli DNA/tube, respectively. PSR detection limits for artificially contaminated pork samples without enrichment, with 12 h enrichment and after 24 h enrichment were 1000 CFU/g, 100 CFU/g, and 10 CFU/g samples, respectively which were ten times better than real-time PCR. The detection performance of PSR (with 12 h enrichment) was also compared to culture (ISO10272-1:2017) method using 75 naturally-contaminated samples, which revealed the sensitivity, specificity, PPV, NPV and accuracy of 100% (95%CI, 73.2%–100%), 98.4% (95%CI, 90%–99.9%), 93.3% (95%CI, 66%–99.6%), 100% (95%CI, 92.5%–100%) and 98.7% (95%CI, 92.8%–99.9%), respectively. The advantage and novelty of this assay are its equipment-free nature, dye-based interpretation by the naked eye, and the requirement of one enzyme and one primer pair. This assay could be a better alternative to other molecular methods and may help in reducing the possible troubles (e.g., gastroenteritis, hospitalization, or death) of belated detection of C. coli in food products. This is the primary report applying the PSR for C. coli detection. [Display omitted] • PSR assay was developed to detect Campylobacter coli for the first time. • The assay involved simple 3-step centrifugation based DNA extraction method. • The assay involved pre-addition of HNB dye for result interpretation. • Analytical sensitivity and detection limit was 62 fg DNA/tube and 100 CFU/g pork. • The assay is equipment-free and requires single enzyme and a primer pair. [ABSTRACT FROM AUTHOR]

For the first time, we describe an innovative polymerase spiral reaction (PSR) assay for the rapid, simple, and accurate detection of pig tissues or pork in adulterated meat including heat-treated and processed ones. The PSR assay specifically targeting the mitochondrial cytochrome b (cyt-b) gene of the pig was successfully optimized permitting assay results in 65 min time. The developed detection method was 100% specific amplifying only the cyt-b gene and displaying negative results with all the tested non-pork meats. The sensitivity of the developed PSR (760 fg porcine DNA) was tenfold better than the end-point PCR and able to detect heat-treated (121 °C) and adulterated (0.5% pork in beef) meat and processed pork products such as sausages, salami, meatball, soup, curry, etc. The developed PSR-based method can be used for point-of-care detection with minimum instrumentation and technical expertise to guarantee instant clearance of exported and imported meat products. This is the first time that PSR has been adapted for food authenticity purposes. [ABSTRACT FROM AUTHOR]

Clostridium perfringens (C. perfringens), a prolific toxin-producing anaerobe is an important foodborne pathogen with a huge public health concern. Rapid and on-site detection of C. perfringens is of specific importance in developing countries. In the present study, saltatory rolling circle amplification (SRCA) assay was developed for culture-independent, rapid and visual detection of C. perfringens and evaluated in meat with pork as a model. The specificity of the SRCA assay was ascertained by using 62 C. perfringens and 18 non- C. perfringens strains. The analytical sensitivity of the developed SRCA, conventional and real-time PCR assays were 80 fg, 800 fg and 800 fg DNA per tube, respectively. The limit of detection of the SRCA assay was 80 CFU/g of pork in the absence of enrichment and 8 CFU/g after short enrichment of 6 h. The detection limits of 80 CFU/g and 8 CFU/g of pork were attained within 120 min and 8 h, respectively. Real-world or field relevancy of the developed assay was evaluated by screening 82 raw and processed pork samples. As the developed assay is simple, user-friendly, cost-effective and sophisticated-equipment free, it would be more suitable for on-site testing of C. perfringens in foods. To our information, this is the first report to apply SRCA for the detection of C. perfringens. [Display omitted] • A novel SRCA assay was developed for rapid and visual detection of Clostridium perfringens in meat. • The analytical sensitivity of the SRCA assay (80 fg) was 10 fold more than the conventional and real-time PCR. • The detection limit of 80 CFU and 8 CFU per gram of meat was attained within 120 min and 8 h, respectively. • This is the first report to use SRCA for the detection of C. perfringens. [ABSTRACT FROM AUTHOR]

The polymerase spiral reaction (PSR), a novel isothermal method for targeted DNA amplification, was effectively applied to detect Salmonella in artificially spiked pork. The specificity of the developed PSR was tested using 16 Salmonella and 15 non- Salmonella strains. The PSR assay was 10-fold more sensitive than conventional end-point PCR, having a sensitivity comparable to real-time PCR. The limit of detection of the developed assay was 4 × 103 per gram of pork without enrichment and 4 CFU per gram after a 6 h enrichment. The detection of 4 CFU per gram of pork was achieved within 8 h. The PSR assay was successful, and accurate in comparison to microbiological methods, in detecting Salmonella in 11 of 76 commercial pork samples. Therefore the positive predictive value, negative predictive value and accuracy rate of the developed assay were 100%. Considering its rapidity, user-friendliness, simplicity, cost-effectiveness and equipment-free nature, this PSR assay is a promising tool for the food industry for the detection of Salmonella and prevention of Salmonella outbreaks and recalls. • Developed a PSR assay for detecting Salmonella in pork and pork products. • The analytical sensitivity of the PSR assay (100 fg) was 10 fold more than the conventional PCR and was comparable to real-time PCR. • The detection of limit of 4 CFU per gram of pork was achieved within 8 h. • Positive predictive value, negative predictive value and accuracy rate of the developed assay was 100%. [ABSTRACT FROM AUTHOR]