BVDV 1型和2型焦磷酸测序检测方法的建立

Development of RT-PCR pyrosequencing for Rapid Detection and typing of BVDV 1 and BVDV 2

为了建立BVDV 1型和2型焦磷酸测序检测方法,用于BVDV的确诊和分型。根据GenBank中发表的BVDV 5'-UTR序列,设计通用扩增引物和分别针对BVDV 1型和2型的两条测序引物。以BVDV 1型BA株和BVDV 2型分离株为模板,用通用引物进行RT-PCR扩增,用测序引物对扩增产物进行焦磷酸测序,建立BVDV 1型和BVDV 2型焦磷酸测序检测方法。结果显示,焦磷酸测序技术检测的序列为30个碱基以上,可用于BVDV的快速鉴定和分型。用该方法对奶牛场86份抗凝血样品进行检测。共检测出2份BVDV 1型,没有检测出BVDV 2型。将检测为阳性的RT-PCR产物测序,测序结果与焦磷酸测序结果一致,符合率为100%。表明本研究建立的BVDV 1型和2型焦磷酸测序检测方法,可用于BVDV的检测和分型,能一次性对待检样品进行确诊,是一种简便、快速的BVDV分型检测方法,对于BVDV的快速确诊具有重要意义。

A pyrosequencing assay based on one-step reverse transcriptase-polymerase chain reaction （RT-PCR） was developed for detection and type of bovine viral diarrhea viruses （BVDV） in genotype 1 and 2. A pair of amplifying primers and sequencing primers used to type BVDV 1 and 2 were designed in the 5-untranslated region of the viral genome. The reverse amplifying primer was labeled with biotin. The BA strain （BVDV 1） and isolated strain （BVDV 2） were used as templates and positive control in the research. The pyrosequencing assay which test more than 30 bp database could detect and type the BVDV rapidly. Test on 86 clinical samples from dairy showed 2 BVDV 1 positive samples. We conclude that the pyrosequencing assay we established is a rapid, specific, high flux and high accurate assay for the detection of BVDV. It can be used to type BVDV status and BVDV epidemiological investigation.