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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]