巴泰病毒实时荧光定量PCR检测方法的建立

Development of a TaqMan Real-time PCR assay for detection of Batai virus

目的利用Taq Man实时荧光定量PCR技术,建立巴泰病毒(Batai virus,BATV)实时荧光定量PCR检测方法。方法根据Gen Bank发表的BATV基因序列资料分析结果,在其S片段编码非结构蛋白的基因区段设计BATV特异引物与探针,利用不同科、属的13株病毒核酸验证方法特异性,利用体外转录的定量RNA标准品建立基因拷贝数定量分析模型,重复实验检验方法的稳定性。结果引物与探针具有良好的特异性,定量分析模型的灵敏度为10拷贝/μl,同一样品重复检测循环阈值的变异系数均<2.50%。结论建立了一种特异、灵敏、高效的BATV一步法Taq Man实时荧光定量PCR检测方法,为今后该病毒的检测与研究工作提供技术手段。

Objective To develop a rapid and sensitive detection method for Batai virus （BATV） based on TaqMan Real-time PCR. Methods Based on the BATV NS gene sequences of S segment published in GenBank, BATV specific primers and probe were designed. The specificity and stability of the system were evaluated. Quantitative standard curve of BATV TaqMan Real-time PCR was established. Results The specificity and stability test showed that the system is specific and the coefficient variables were all less than 2.50%. Quantitative standard curve based on the genomic copy was drawn, and the lowest detectable limit （LOD） of system is 10 copies/μl. Conclusion TaqMan Real-time PCR for BATV detection has been developed, which is more sensitive and more efficient than the general PCR.