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为建立同时快速检测猪A群轮状病毒(GARV)、猪C群轮状病毒(GCRV)和猪星状病毒(AstV)的三重RT-PCR检测方法,根据GenBank中公布的序列进行病毒基因区同源性分析,然后使用Primer Premier 5.0软件分别设计了GARV、GCRV的VP6基因和AstV的ORF2基因特异性引物,预期扩增片段的大小分别为229、166和410bp。在这3种病毒单项RT-PCR技术的基础上,建立了GARV、GCRV和AstV的多重RT-PCR检测方法,并用该方法检测30份四川省仔猪腹泻粪样。结果显示,GARV、GCRV、AstV的阳性检出率分别为26.7%、13.3%、16.7%,本方法与这3种病毒的单项RT-PCR检测结果的符合率分别为100%、100%、83%,整体符合率达94.3%,特异性为100%。结果表明,本试验建立的多重RTPCR检测方法具有特异、快速、准确的特点,可用于GARV、GCRV和AstV的同时检测。
In order to establish a triple RT-PCR method for rapid simultaneous detection of porcine a-swine rotavirus (GARV), porcine C-group rotavirus (GCRV) and swine vesicular virus (AstV), the viral gene region Homology analysis and then using the Primer Premier 5.0 software were designed GARV, GCRV VP6 gene and AstV ORF2 gene-specific primers, the expected size of the amplified fragments were 229,166 and 410bp. Based on the three single-nucleotide RT-PCR techniques, multiple RT-PCR methods for detecting GARV, GCRV and AstV were established, and 30 samples of diarrhea in Sichuan piglets were detected by this method. The results showed that the positive rates of GARV, GCRV and AstV were 26.7%, 13.3% and 16.7%, respectively. The coincidence rates of this method with the single RT-PCR results of these three viruses were 100%, 100%, 83 %, The overall compliance rate of 94.3%, specificity of 100%. The results showed that the multiplex RTPCR assay established in this study was specific, rapid and accurate and could be used for the simultaneous detection of GARV, GCRV and AstV.