通用型猪流感病毒PCR-DHPLC检测方法的建立

Development of PCR-DHPLC for universal detection of swine influenza virus

应用PCR技术结合变性高效液相色谱(DHPLC)技术,建立通用型猪流感病毒的快速筛检方法。查找、分析猪流感病毒M基因的保守区,设计了1对特异性引物。RT-PCR扩增后经变性高效液相色谱技术分析研究方法的特异性、敏感性和应用性。DHPLC法能特异性检出H1N1、H5N1和H3N2亚型猪流感病毒,与猪瘟病毒、口蹄疫病毒、猪流行性腹泻病毒、猪繁殖与呼吸综合征病毒、猪圆环病毒2型无交叉反应。对构建的含有M基因的重组质粒,该方法的检出下限为100copies核酸。对210份存档猪流感鼻拭子样品,DHPLC法与商业化定量PCR的检测结果完全一致。DHPLC技术为临床大批量猪流感样品的监测提供了一种新的、自动化、高通量的核酸分析平台。

In order to detect swine influenza virus(SIV),a new method was established using RT-PCR and denaturing high performance liquid chromatography(DHPLC).According to the conserved region of matrix gene of SIV,one pair of primers was designed.After being amplified,PCR products were analyzed by DHPLC.When H1N1,H5N1,and H3N2 SIV subtypes were detected by DHPLC,no positive absorbtion peaks occurred with classical swine fever virus,foot-and-mouth disease virus,porcine epidemic diarrhea virus,porcine reproductive and respiratory syndrome and porcine circovirus type 2.A detection limit of PCRDHPLC method was 100 copies recombinant plasmids.A total 210 archived SIV samples were tested by PCR-DHPLC and real-time PCR,showing 100%agreement.The result showed that PCR-DHPLC is an automatic and high throughput method for detection of clinical samples.