SYBR Green Ⅰ荧光定量PCR法检测猪流感H3N2亚型病毒方法的建立

Establishment of SYBR Green Ⅰ real time PCR for detection of swine influenza virus H3N2 subtype

本试验针对H3N2亚型流感病毒神经氨酸酶基因建立SYBR GreenⅠ荧光RT-PCR检测方法。根据GenBank上的SIV N2基因序列设计一对引物,扩增片段406bp。纯化扩增目的片段后连接pMD18-T载体中,命名为pMD18-TN2,转化入E.coli DH5α,测序正确作为阳性模板,优化反应条件建立方法。此荧光PCR方法所能检测最低病毒含量为75copies/μL,拥有良好的特异性和重复性。此方法的建立将为流感病毒N2亚型神经氨酸酶基因定型检测提供一种有效方法。

This experiment set up a real time PCR（FQ-PCR） with SYBR Green Ⅰ as fluorescent dyes which could detect the Neuraminidase gene of swine influenza virus H3N2 subtype.According to conservative sequence of N2 gene design a pair of primers.Using this pair of primers to amplify and purify N2 gene.Then clone N2 DNA fragment into pMD18-T vector.Named it pMD18-TN2.Used pMD18-TN2 as positive control to create and optimize the reaction conditions.The results show that the lowest content cDNA of SIV that the SYBR Green Ⅰ real-time PCR can detect is 75 copies/μL,and it also has good specificity and reproducibility.The establishment of this assay will provide an effective method for detecting Neuraminidase gene of swine influenza virus H3N2 subtype.