A 群轮状病毒, 塞内卡病毒, 双重 RT-qPCR 检测方法建立及应用

To establish a dual RT-qPCR detection method for porcine rotavirus A ( PoRVA) and seneca virus A ( SVA) and conduct preliminary applications. Methods By comparing the sequences of PoRVA and SVA strains, conserved regions of the PoRVA NSP3 gene and SVA VP1 gene were selected to design primers and probes, respectively, and a dual RT-qPCR detection method for PoRVA and SVA was established. Subsequently, the specificity and sensitivity of the method was confirmed.Recombinant plasmid standards with concentrations ranging from 10 6 to 10 3 copies/ μL were serially diluted 10 - fold. Each concentration was tested in triplicate and the tests were repeated three times.Intra- and inter-assay coefficients of variation ( CV) were calculated to evaluate the repeatability and stability of the method. The established dual RT-qPCR method was compared with the detection method recommended by national standard “ GB / T 34756 - 2017” and industry standard “ SN / T 5486 - 2022” .Additionally, 15 pig fecal samples and 35 pig anal swab samples were tested to evaluate the practical applicability of the method. Results The established dual RT-qPCR method showed a linear relationship between Ct values and plasmid concentrations in the range of 109-101copies/ μL. The standard curve for PoRVA had a slope of -3. 085, an R 2 value of 0. 993, and an amplification efficiency of 110. 956%. The standard curve for SVA had a slope of -3. 085, an R 2 value of 0. 993, and an amplification efficiency of 110. 935%. The minimum detectable concentrations for both PoRVA and SVA were 101 copies/ μL. No cross-reactivity was observed with other seven common swine pathogens. Repeated testing of plasmid standards at concentrations of 106 to 103 copies/ μL showed intra-assay CVs of less than 0. 15% and inter assay CVs of less than 1. 50%. The established dual RT-qPCR method and the national standard and industry standard method were used to detect the positive rate of 50 samples. The result showed that the positive rate of PoRVA and SVA were 100% (50 / 50) and 66% (33 / 50) for the dual RT-qPCR method, respectively. The positive rate of PoRVA and SVA were 100% (50 / 50) and 24% ( 12 / 50) for “GB / T 34756- 2017” and “ SN / T 5486 - 2022” method, respectively. The positive concordance rate reached 100%. Conclusion The dual RT-qPCR detection method established in this study can effectively detect PoRVA and SVA, providing technical support for disease detection and routine monitoring in swine populations.

Porcine rotavirus A, Seneca virus A, establishment and application of a duplex RT-qPCR detection method