H1亚型猪流感病毒一步法实时荧光定量RT-PCR检测方法的建立及应用

Abstract

Abstract: To establish a rapid, simple and accurate method to diagnose and detect H1 subtype swine influenza virus (SIV), the specific primers and TaqMan MGB probe were designed according to the conserved region of HA gene of H1 subtype SIV. A one-step Real-time fluorescent quantitative RT-PCR assay was developed for detection of H1 subtype SIV. A series of dilutions of recombinant plasmids pMD18-H1 were prepared and used to generate standard curves. The results showed that the one-step Real-time fluorescent quantitative RT-PCR was capable of detecting 10² copies of H1 subtype SIV per microliter. The results were negative for the detection of H3N2 and H9N1 subtypes SIV, classical swine fever virus, porcine reproductive and respiratory syndrome virus, porcine epidemic diarrhea virus, transmissible gastroenteritis virus. The coincidence rates between one-step Real-time fluorescent quantitative RT-PCR and virus isolation were 94%. The method was highly specific and sensitive, and could be used for rapid quantitative detection of H1 subtype SIV.

Key words: swine influenza virus; H1 subtype; TaqMan MGB probe; one-step Real-time fluorescent quantitative RT-PCR

CLC Number:

LIU Hao-peng, HU Jing-jing, TANG Xu, PEI Zhang-fu, LI Bing, LI Lin, CHEN Rui-ai, HE Dong-sheng. Development and Application of One-step Real-time Fluorescent Quantitative RT-PCR Assay for Rapid Detection of H1 Subtype Swine Influenza Virus[J]. , 2014, 41(11): 63-68.

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Development and Application of One-step Real-time Fluorescent Quantitative RT-PCR Assay for Rapid Detection of H1 Subtype Swine Influenza Virus

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