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Methicillin resistant Staphylococcus aureus harbouring other virulence factors in food is a serious threat to food safety. Delayed detection of MRSA may lead to fatal consequences due to the challenging treatment methods. To address the issue, early prognosis is prerequisite. We report one step QuEChERS detection method wherein, a media with selective agents such as cefoxitin, cyanidin, lithium chloride and methicillin (CCLM) was formulated for the specific cultivation and detection of MRSA within 5 hours. Direct application of the newly developed method was tested by screening food for MRSA and its comparison with conventional biochemical and molecular characterization. Growth of MRSA in the CCLM media was observed within 4 hours. This highly specific method is sensitive to detect 104 CFU/mL visually and 10¹ CFU/mL spectrophotometrically. 25% of food samples harboured MRSA which points to an immediate need of consideration by the authorities. The newly designed QuEChERS cyanin based chromogenic detection system is unequivocal with the conventional and molecular method of MRSA detection and can be of great use in diagnostic and in food safety laboratories. [ABSTRACT FROM AUTHOR]

Cytolethal distending toxin ( CDT) producing Campylobacter jejuni species are one of the leading causes of human gastroenteritis worldwide. The main intent of the study was to develop a multiplex PCR assay for the confirmed identification and toxin profiling of C. jejuni. The genes targeted were rpo B as genus specific, hip O for species; cdt A, cdt B, cdt C encoding respective subunit proteins of CDT with Internal Amplification Control ( IAC). To enhance its application as a pre-mixed ready-to-use format, the master mix of developed mPCR was dried by lyophilization and stability was assessed. Thermostabilized reagents showed stability of 1.5 months at room-temperature and upto six months at 4 °C without any loss of functionality. The assay was evaluated on a number of presumptive Campylobacter isolates along with biochemical tests. Results obtained indicated the accurate identification of C. jejuni by developed mPCR format in contrast to misconception associated with biochemical assays. The assay was also tested on spiked samples for its real-time utility. Altogether, the room-temperature storable and ready-to- use mPCR format developed in this study could be preferred for rapid detection and confirmed identification of toxigenic strains of C. jejuni in place of conventional biochemical assays. [ABSTRACT FROM AUTHOR]

In spite of their involvement in foodborne illness, the epidemiological relevance of staphylococcal enterotoxin C (SEC) subtypes is poorly documented may be due to high sequence similarity. Among subtypes, SEC1, SEC2, and SEC3 exhibit more than 97 % homology because of which specific detection tools are seldom available to identify and differentiate them . In this study, a SYBR Green-based RT-PCR followed by melt curve analysis was developed for differentiation of entC1 from entC2/ entC3 using a single primer pair. Nucleotide sequences of all three subtypes were analyzed using Clustal Omega program and the region with significant sequence variation/heterogeneity (where utmost SNPs were closely located and accessible for RT-PCR) was selected for amplification by designing a single primer pair that could amplify all three subtypes. In spite of same amplicon size, entC1 showed distinct melt peak at 76 °C. However, due to high similarity between entC2 and entC3, the developed format was deficient to discriminate between them and both showed melt peak at 82 °C. Reliability of developed RT-PCR was evaluated using various naturally contaminated samples and 91 food and clinical Staphylococcus aureus isolates where satisfactory results were obtained in comparison with commercial immunoassay kit and conventional PCRs using validated primers. To the best of our knowledge, this is the first method being reported to differentiate entC1 from entC2/ entC3 using single primer pair which is unachievable by conventional PCR due to same amplicon size. As benefits, the method is sensitive, rapid, and inexpensive with no requirement of fluorescent probes, multiple primers, and post-PCR procedures. Thus, the assay might find its utility as a detection tool in epidemiological survey of foodborne outbreaks for simultaneous identification and differentiation of entC1 from entC2/ entC3. [ABSTRACT FROM AUTHOR]