H3亚型猪流感病毒SYBR GreenⅠ实时荧光定量PCR检测方法的建立

Development of a SYBR Green Ⅰ real-time fluorescent quantitative PCR assay for detection of swine influenza virus H3 subtype

为建立一种快捷、可行的检测H3亚型猪流感病毒(SIV)的方法,本研究根据GenBank中登录的H3亚型SIV HA基因的保守序列,设计合成一对特异性引物,建立了一种检测H3亚型SIV的SYBR GreenⅠ荧光定量检测方法,并进行灵敏度、稳定性和特异性试验,与普通PCR进行比较。研究结果表明,标准曲线的循环阈值与模板浓度呈现良好的线性关系,R2为0.994,CV在0.17%～1.41%之间,具有良好稳定性。除H3亚型SIV外,对H1、H4、H6、H9亚型SIV以及猪瘟病毒、猪繁殖与呼吸综合征病毒、口蹄疫病毒和猪圆环病毒2型的检测均为阴性,与普通PCR相比更灵敏。该方法特异性好,准确率高,适于临床分离鉴别H3亚型SIV。

In this study, a SYBR Green I real-time PCR assay was developed for detection of swine influenza virus （SIV） subtype H3 with the specific primers designed according to the conserved sequence of HA genes of SIV tt3 subtype in Gcnl3ank. The results showed that the standard curve established by recombinant plasmid showed a good linear relationship between templateconcentration and threshold cycle, the correlation coefficient （R^2） was 0.994 and the coefficient of variation （CV） of intra-assay was between 0.17% and 1.41%. No cross-reaction was detected for other swine viruses and HI, H4, H6 and H9 subtypes of SIV. The results indicated that real-time PCR is more sensitive than routine PCR and virus isolation which was a promising application in diagnose of clinical suspected cases.