牛副流感病毒3型实时荧光定量PCR检测方法的建立及在牛源样本检测中的应用

Establishment and application in bovine origin samples of real-time fluorescent quantitative PCR method for the detection of bovine oarainfluenza virus 3

目的建立用于牛源性样本牛副流感病毒3型(BPIV3)实时荧光定量PCR (Q-PCR)快速检测方法。方法选择已发表的BPIV3 HN基因高度保守区域作为靶基因,设计合成引物和探针,建立BPIV3Q-PCR方法。并对方法的线性、特异性、敏感性、重复性、稳定性等进行方法学评价。同时用建立的Q-PCR方法检测105批次牛源性样本。结果建立的BPIV3Q-PCR方法线性范围为1×101拷贝/μl^1×108拷贝/μl;与牛病毒性腹泻病毒(BVDV)、牛鼻气管炎病毒(BHV-1)、仙台病毒(SV)均无交叉反应;检测敏感度可以达到1.0×10~1拷贝/μl;3个不同浓度梯度标准品3次不同实验平均变异系数均小于5%。应用建立的方法检测105批次牛源性样本,BPIV3核酸阳性率为12.4%。结论建立的BPIV3QPCR检测方法线性关系良好,具有快速、特异、敏感及稳定的特点,可用于牛源性样本BPIV3的快速定量检测。

Objective To establish a real-time fluorescent quantitative PCR（Q-PCR）method for the detection of bovine parainfluenza virus 3（BPIV3）in bovine origin samples. Methods The PCR primers and TaqMan probe were designed and synthesized according to the published BPIV3 specific sequences of HN gene.Q-PCR method was established,and the linearity,specificity,sensitivity,stability of this method were evaluated.Then,the method was used to detect 105 bovine origin samples. Results The linear range was101 copies/μl to 108 copies/μl.The developed Q-PCR method showed no cross reactions with bovine viral diarrhea virus（BVDV）,bovine herpesvirus type1（BHV-1）,and sendai virus（SV）.The limit of detection was1.0×10-1 copies/μl.Coefficient of variation（CV）was less than 5%.The nucleic acid positive rate of BPIV3 was 12.4% detected by this Q-PCR method. Conclusions The developed Q-PCR method is good in linearity,specificity,sensitivity,stability,and can be used for the rapid quantitative detection of BPIV3 in bovine origin samples.