H9N2亚型禽流感病毒荧光定量PCR方法的建立及初步应用

摘要

H9N2亚型禽流感病毒（Avian influenza virus, AIV）是我国主要流行的低致病性禽流感病毒亚型，因而对其进行病毒的监测与诊断显得极其重要。通过比对GenBank上H9亚型禽流感病毒HA基因序列,设计了一个特异性针对H9 AIV HA的探针。特异性和扩增曲线实验结果显示，该探针具有较好的扩增效率，且仅特异性识别H9亚型AIV。通过重组质粒pMD19-T-H9HA 10倍梯度稀释液为模板，进行荧光定量PCR。实验结果表明，本研究所建立的荧光定量PCR方法敏感性能达到100个DNA拷贝数，比传统PCR方法检测敏感性提高了1000倍。此外，本方法还能检测到10EID50个病毒，比传统PCR方法检测敏感性也提高了1000倍。通过批间和批内试验，结果表明，这两个试验的变异系数分别小于5%和1%，说明本研究的方法具有较好的重复性。此外，通过检测人工感染动物咽拭子，结果表明H9亚型AIV荧光定量PCR方法比传统PCR的方法检测敏感性提高了100倍。在临床样品的检测中，H9亚型AIV荧光定量PCR方法检测的阳性率比传统PCR法检测的阳性率提高了大约30%。综上所述，本研究所建立的H9N2亚型禽流感病毒荧光定量PCR方法具有较高的特异性、准确性和敏感性，对H9亚型禽流感疫情防控及其流行病学调查具有积极的科学意义。%　　H9N2 Avian influenza virus (AIV) is a major subtype with low pathogenecity in China. For surveillance and diagnosis of H9N2 AIV, we designed TaqMan primers based on HA gene sequences of H9 strains deposited in the GenBank. Preliminary trials demonstrated that these primers were specific for H9 AIV and induced efficient amplification. The quantitative PCR (qPCR) was performed using a series of 10-fold dilutions of pMD19-T-H9HA as templates. The qPCR was able to detect 100 DNA copies, which was 1000-fold higher than conventional PCR. In addition, qPCR also detected 10 EID50 viruses, 1000-fold higher than conventional PCR. The variability of inter-plates and intra-plates were less than 5%and 1%, respectively. Then, qPCR was used to detect oropharyngeal swab samples. As a result, qPCR was 100-fold more sensitive than conventional PCR in testing clinical samples. Therefore, qPCR method was highly specific, accurate, reproducible and sensitive and thus could be used for diagnosis of H9N2 AIV.