牦牛轮状病毒VP6基因序列分析及RT-PCR检测方法的建立与应用  

Establishment and Application of an RT-PCR Assay for Yak Rotavirus Based on the Sequence Analysis of Yak Rotavirus VP6 gene

作　　者：周芳[1] 岳华[1] 张斌[1] 李凡[1] 陈曦[1] 汤承[1] 

机构地区：[1]西南民族大学生命科学与技术学院/四川省教育厅重大动物疫病防控技术创新团队,成都610041

出　　处：《畜牧兽医学报》2016年第7期1465-1473,共9页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金："十二五"科技支撑计划项目(2012BAD13B06);四川省科技计划项目青年基金(2014JQ0044)

摘　　要：轮状病毒(rotavirus,RV)VP6基因是RV的主要分子检测靶点,但该基因的点突变会影响RV的分子检测。本试验的目的是在研究牦牛RVVP6基因遗传变异的基础上,建立检测牦牛RV的RT-PCR方法并应用于临床样本检测。采用Prime 5.0设计引物,从30个牦牛腹泻样本中扩增得到14个位于931—1 338bp牦牛RVVP6基因片段。序列分析结果发现,与牛轮状病毒(bovine rotavirus,BRV)相比,牦牛RVVP6基因在931—1 110bp间出现多处点突变,而在1 100—1 338bp之间序列高度保守。据此设计检测引物,成功建立了检测牦牛RV的RTPCR方法,具有良好的特异性和稳定性,只扩增出牦牛RV及BRV的特异片段,对其他无关病原无扩增;检测下限为0.45pg·μL^(-1),灵敏性较好。所建立方法对牦牛RV的检出率明显优于现有检测BRV的RT-PCR方法,为牦牛RV病的诊断和流行病学调查提供了可靠的方法;对BRV的检出也与其他方法具有很好的符合率,也可以用于BRV的检测。对234份犊牦牛腹泻粪便样本的RV检出率:西藏为60.00%(36/60)、青海为95.00%(57/60);四川为85.19%(46/54);云南为90.00%(54/60),证明牦牛RV感染是当前导致犊牦牛腹泻的重要原因。Rotavirus（RV）VP6gene is the main target gene of molecular detection,but the gene point mutations affects the molecular detection of RV.The purpose of this experiment was to establish a detection method of yak RV by RT-PCR method which applied in clinical samples based on the study of genetic variation yak RVVP6.The fragments of yak RVVP6 ranged in 931-1 338 bp were obtained with the primers designed by Prime 5.0software from 30 samples of yak diarrhea.Sequence analysis showed that,yak RVVP6 genes represented multiple point mutations in the section of 931-1110 bp compared with bovine rotavirus（BRV）,while the section between1 100-1 338 bp was highly conserved.According to this result,the assay has successfully established a method with good specificity and stability.The primer could amplify the specific fragment of yak RV and BRV,with no amplification of other unrelated pathogens.The detection limit of viral nucleic acid of the assays was 0.45pg·μL^（-1）.With a remarkable detection rate in yak RV,the RT-PCR method provided a useful tool for the diagnosis and epidemiological investigation of yakRV disease.Additionally,this method can also be used in BRV detection.The RV detection rate in234 samples of yak diarrhea from different provinces were 60.00%in Tibet（36/60）,95.00%in Qinghai（57/60）,85.19%in Sichuan（46/54）and 90.00%in Yunnan（54/60）,respectively.The result shows that RV infection is an important cause of yak diarrhea.

关 键 词：牦牛 轮状病毒 VP6基因 点突变 RT-PCR 

分 类 号：S852.653[农业科学—基础兽医学] S852.659.3[农业科学—兽医学]