Abstract:
Objective A rapid method for detecting porcine rotavirus group A (PoRV A) in disease monitoring and epidemiological investigation was developed.
Method Specific primers and probes were selected based on the VP6 gene sequences of PoRV A in GenBank (accession numbers MT025937.1, OP978242.1 and PP566178.1). Reaction conditions of the TaqMan RT-qPCR method were optimized followed by evaluations on assay specificity, sensitivity, repeatability, and clinical application.
Results The newly developed method could specifically amplify PoRV nucleic acid with a minimum detection limit of 27.0 copies·μL−1 and a sensitivity 100 times higher than RTPCR. There was no cross reactivity with the nucleic acids of PEDV, TGEV or PDCoV. The method showed high repeatability with the variation coefficients on intra- and inter-group below 1.10%. On 151 clinical specimens suspected of PoRV, a detection rate of 42.38% (64/151), which was better than that of conventional RT-PCR at 33.11% (50/151), was obtained by the assay.
Conclusion The new TaqMan RT qPCR method for detecting VP6 gene of PoRV A was high in assay sensitivity, specificity, and repeatability. It was considered suitable for PoRV detection and epidemiological investigations.
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