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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Authors: | LIU Hao-peng HU Jing-jing TANG Xu PEI Zhang-fu LI Bing LI Lin CHEN Rui-ai HE Dong-sheng |
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Institution: | 1. Guangdong Dahuanong Animal Health Product Co., Ltd., Xinxing 527400, China;2. College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China |
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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. |
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Keywords: | swine influenza virus H1 subtype TaqMan MGB probe one-step Real-time fluorescent quantitative RT-PCR |
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