A Duplex polymerase chain reaction (PCR) was developed for the simultaneous detection and differentiation among Nosema spp. and Vairimorpha spp. from Lepidoptera insects. Two sets of primers were selected from different genomic sequences to specifically amplify an 831 bp amplicon within the SSU rRNA gene, specific for both Nosema spp. and Vairimorpha spp. (MSSR primer); a 542 bp amplicon within the SSU rRNA gene, specific for Vairimorpha spp. (VSSU primer). Using the primers in conjunction (duplex PCR) it was possible to detect Nosema spp. and Vairimorpha spp. to differentiate between them. The sensitivity of this PCR assay was approximately 10 spores per milliliter. It is proposed that the duplex PCR is a sensitive, specific and rapid tool that can serve as a useful differential diagnostic tool for detecting Nosema spp. and Vairimorpha spp. in Lepidoptera insect.

In this study, two duplex real-time PCR approach with melting curve analysis is presented for the detection of Escherichia coli O157:H7, Listeria monocytogenes, Salmonella spp. and Staphylococcus aureus, which are important food-borne bacterial pathogens usually present in fresh and/or minimally processed vegetables. Reaction conditions were adjusted for the simultaneous amplification and detection of specific fragments in the β-glucuronidase (uidA, E. coli), thermonuclease (nuc, S. aureus), hemolycin (hly, L. monocytogenes) and tetrathionate reductase (ttr, Salmonella spp.) genes. Melting curve analysis using a SYBR Green I real-time PCR approach showed characteristic Tm values demonstrating the specific and efficient amplification of the four pathogens; 80.6 ± 0.9 ℃,86.9 ± 0.5 ℃, 80.4 ± 0.6 ℃ and 88.1 ± 0.11 ℃ for S. aureus, E. coli O157:H7, L. monocytogenes and Salmonella spp.,respectively. For all the pathogens, the two duplex, real-time PCR was equally sensitive to uniplex real-time PCR,using same amounts of purified DNA, and allowed detection of 10 genome equivalents. When our established duplex real-time PCR assay was applied to artificially inoculated fresh lettuce, the detection limit was 10³ CFU/g for each of these pathogens without enrichment. The results from this study showed that the developed duplex real-time PCR with melting curve analysis is promising as a rapid and cost-effective test method for improving food safety.