我国口蹄疫流行血清型RT-PCR鉴别诊断技术的建立

RT-PCR technique for determining the prevalent serotypes of the foot and mouth disease virus in China

目的建立口蹄疫流行株不同血清型RT—PCR的鉴别诊断技术，为有效防控口蹄疫提供技术支撑。方法根据GenBank中的A型、0型、Asial型口蹄疫病毒的全基因组核酸序列，用Primer5．0设计该3型口蹄疫病毒的通用引物及特异性分型引物，建立RT—PCR方法，采用1％琼脂糖凝胶电泳结合测序技术对引物的特异性和广谱性进行分析。结果1％琼脂糖电泳结果表明通用引物P1／P2能同时特异性扩增A、O、Asial型口蹄疫病毒，扩增片段大小为457bp，与预期值相一致。A、O、Asial型分型引物只能扩增相应亚型口蹄疫病毒的目的基因片段（大小分别为320、240和460bp），与预期值相一致。不能扩增其他2种亚型。Blast比对显示分型引物扩增产物序列经测序验证与其亚型完全对应，同源性为84％～99％。结论建立的RT—PCR诊断技术对口蹄疫病毒通用型扩增具有广谱性，对基因亚型分型具有特异性，可用于口蹄疫病检测及其流行病学调查。

Objective The aim of this study was to establish an RT-PCR technique for determining the prevalent sero- types of the foot and mouth disease virus in China in order to provide technical support for the prevention and control of foot and mouth disease. Methods The entire genome of types A, O, and Asial of the foot and mouth disease virus in GenBank was compared, and Primer 5.0 was used to design universal primers and specific primers for types A, O, and Asia1. One percent agarose electrophoresis and sequencing were also used to analyze the serotypes of the foot and mouth disease virus. Results One percent agarose electrophoresis indicated that the universal primers P1/P2 amplify types A, O, and Asia1, yielding fragments 457 bp in length. Specific primers for types A, O, and Asia1 amplified only subtypes of the corresponding type and did not amplify subtypes of the other 2 types. Yielded fragments were 320 bp, 240 bp, and 460 bp in length. Sequencing and identification completely accorded with the results of RT-PCR amplification. Similarity was 84 %- 99 %. Conclusion An RT-PCR technique was established to determine the serotypes of the foot and mouth disease virus. This technique universally amplified fragments of the virus and it specifically identified different subtypes. These findings have laid the foundation for testing of clinical samples and epidemiological investigation of foot and mouth disease.