牛病毒性腹泻病毒基因分型二重RT-PCR检测方法的建立

Establishment of a RT-PCR assay for simultaneous dutecion of BVDV

为了同步检测牛病毒性腹泻病毒(BVDV)基因1型和基因2型并能够加以区分,根据Gen Bank中已发表的BVDV基因1型和基因2型、牛蓝舌病病毒(BTV)、口蹄疫病毒(FMDV)、猪瘟病毒(CSFV)、牛传染性鼻气管炎病毒(IBRV)的全基因序列,采用Primer Premier 5.0软件,针对BVDV的高度保守的5'端非编码区的核苷酸序列,设计了两对特异性引物,二重RT-PCR方法扩增目的片段的大小分别为222 bp和119 bp,经过对反应体系和条件的优化,本研究建立了在同一反应体系中鉴别检测BVDV基因1型和2型的RT-PCR方法,扩增出了与预期大小相符的目的片段。该方法便捷,特异性高,敏感性强,重复性好,可作为BVDV分型鉴定和快速诊断的有效方法。

In order to detect and distinguish the genome sequences of genotype 1 BVDV and genotype 2 BVDV simultaneously, two pairs of primers that the specific to the 5＂- untranslated region of BVDV were designed by Primer Premier 5.0, according to the complete sequences of BVDV1, BVDV2, BTV, FMDV, CSFV and IBRV in GenBank. The 5＇-untranslated region of BVDV was the highest conservative region in the BVDV complete genome. The target fragments＇ sizes were 222 bp and 119 bp. After the optimiza- tion of the reaction conditions, we set up a method which can distinguish genotypes of BVDV using RT-PCR just in one reaction sys- tem. This established assay can detect BVDV1 and BVDV2 specifically, sensitively and reproducibly, which is an efficient assay for BVDV detection and genotyping.