鉴别蓝舌病病毒血清29型RT-PCR与qRT-PCR方法的建立与应用

Establishment and application of RT-PCR and qRT-PCR methods for identification of BTV-29

为建立快速鉴别蓝舌病病毒血清29型(BTV-29)的方法,根据BTV-29型毒株基因节段2(Seg-2)的序列特征,分别设计用于RT-PCR与q RT-PCR方法的特异性引物与Taq Man探针,通过试验确定其敏感性,用BTV-1~BTV-24等核酸作为对照评价其特异性。对BTV-29感染动物血液样本中的病毒核酸进行监测,并用BTV群特异q RT-PCR方法验证其可靠性。结果显示,建立的BTV-29特异RT-PCR的灵敏度是1.12×10^(1)copies/μL,而BTV-29特异q RT-PCR的灵敏度是1.12×10^(2)copies/μL,均与BTV-1~BTV-24、流行性出血病病毒、帕利亚姆病毒和阿卡斑病毒之间无交叉反应;用BTV-29特异q RT-PCR检测BTV-29型感染山羊血液中的BTV-29核酸的动态变化,结果与BTV群特异q RT-PCR检测结果基本一致。本研究建立的BTV-29特异RT-PCR和q RT-PCR方法均具有良好的特异性和较高的灵敏度,能快速鉴别BTV-29,为BTV-29的致病性、诊断与流行病学研究提供了技术保障。

To establish the quick detection method for the identification of bluetongue virus serotype 29(BTV-29),BTV-29 specific primers and Taq Man probe for RT-PCR and q RT-PCR were designed,respectively,the sensitivities of two methods were determined,and the specificities were evaluated using the nucleic acids of BTV-1—BTV-24.The blood samples of animals infected with BTV-29 were monitored the viral nucleic acid,and the reliability of the result was verified by BTV group-specific q RT-PCR.The results showed that the sensitivity for nucleic acid of BTV-29 were 1.12×10^(1) copies/μL for RT-PCR and 1.12×10^(2) copies/μL for q RT-PCR,respectively,and BTV-29-specific RT-PCR and q RT-PCR were no cross reaction with the nucleic acids of from BTV-1 to BTV-24,epidemic haemorrhagic disease virus,Palyam virus and Akabanne virus.The detection results of BTV-29 infected goats with BTV-29-specific q RT-PCR was basically consistent with the detection result of the BTV group-specific q RTPCR.The BTV-29 specific RT-PCR and q RT-PCR method established in this study shared good specificity and high sensitivity,and could quickly identify the serotype of BTV-29.The established methods provided technical support for the pathogenicity,diagnosis and epidemiology research of BTV-29.