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Brucellosis is an economically important livestock disease worldwide besides having a noteworthy impact on human health. In this study, a rapid, simple, and ultra-sensitive nuclei-acid diagnostic technique was developed for the detection of brucellosis harnessing saltatory rolling circle amplification (SRCA). The diagnostic method was developed using World Organization for Animal Health (WOAH) approved primers targeting the bcsp31 gene of the Brucella genome. The assay can be accomplished within 90 min at a temperature of 65 °C without the requirement of sophisticated instrumentation. The result interpretation can be done with the naked eye with the aid of SYBR green dye. The developed technique displayed 100% specificity by amplifying only 10 reference and field strains of Brucella spp. and there was no cross-reactivity with the other tested pathogens. The lower limit of detections of SRCA and end-point PCR assays were 9.7 fg/μL (2.7 genome copies of Brucella) and 970 fg/μL, respectively. Thus, the developed SRCA assay was found to be 100× more sensitive than the end-point PCR assay. To the best of our knowledge, our study is the first one to develop an SRCA-based assay for the detection of brucellosis and it can be a diagnostic tool for resource-constrained laboratories and veterinary hospitals. • Saltatory rolling circle amplification technique for the detection of Brucella. • Simple and ultrasensitive diagnostic method for specific detection of Brucella. • Technique operates at isothermal temperature and provides results in 90 min. • Detection limit (9.6 fg/μL) is 100× more sensitive than end-point PCR. [ABSTRACT FROM AUTHOR]

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]

Staphylococcus aureus is one of the most common and leading causes of food poisoning and also responsible for multifarious diseases in humans. Rapid and on-site detection of S. aureus is of specific importance in developing countries. In the present study, a polymerase spiral reaction (PSR) assay was developed for sensitive, rapid and visual detection of S. aureus and validated in meat using pork as a model. The specificity of the PSR assay was ascertained by using 18 S. aureus and 17 non- S.aureus strains. The PSR assay was 100- and 10-fold more sensitive than conventional end-point PCR and real-time PCR, respectively. The limit of detection of the PSR assay was 19.9 × 103 CFU/g of pork without enrichment and 19.9 CFU/g after 6 h enrichment. The detection of 19.9 CFU/g of pork was attained within 8 h. Real-world or field applicability of the developed assay was evaluated by screening 76 raw and processed pork samples. Seeing its rapidity, simplicity, cost-effectiveness, user-friendliness and sophisticated-equipment free nature, this assay has the prospective to turn into the assay of choice for regular detection of S. aureus in foods in less-advanced laboratories. To our knowledge, this is the first report to use PSR for the detection of S. aureus. • A novel PSR assay was developed for rapid and visual detection of Staphylococcus aureus in meat. • The analytical sensitivity of the PSR assay was 100 and 10 fold more than the conventional and real-time PCR, respectively. • The detection of limit of 19.9 × 103 CFU and 19.9 CFU per gram of meat was attained within 2 h and 8 h, respectively. [ABSTRACT FROM AUTHOR]