不同标本中甲型肝炎病毒核酸检测方法的建立及应用

Development of an extraction and concentration method for the detection of hepatitis A virus in different samples

目的建立水、贝类、血液及唾液中甲型肝炎病毒核酸RT—PCR检测方法。方法加入一定量甲肝病毒到贝类、水、血液及唾液标本中，贝类经研磨后，PEG沉淀浓缩；水直接经PEG沉淀浓缩：以上标本及血液、唾液标本用Trizol试剂提取核酸，套式RT-PCR检测甲肝病毒核酸。引物位于甲肝病毒结构区VPI-2A区。结果病毒培养液中能检测到0．1TCID50甲肝病毒；水、血清及唾液中能检测到1TCID50甲肝病毒；毛蚶中能检测到1-10TCID50甲肝病毒。某地甲肝病毒污染的水及患者血清标本中检测到了甲肝病毒核酸，并进行了序列分析比对。结论本研究建立的不同标本中甲型肝炎病毒核酸RT-PCR检测方法，敏感、特异、快速，具有广泛的应用价值，将为甲肝的溯源研究及分子流行病学研究打下理论基础及提供技术支撑.

Objective To develop an extraction and concentration method for the detection of hepatitis A virus （HAV） in shellfish, water, serum and saliva samples by nested RT-PCR. Methods HAV were artificially inoculated into the above samples, calm sample was extracted using glycine buffer pH9.5, PEG precipitation; water sample was PEG precipitated directly; then all the samples including serum and saliva samples were extracted using Trizol regent, followed by nested RT-PCR detection using primers from HAV VPI-2A region. Results The detection limit for HAV in cultured cell lysis was 0.1TCID50; in water, serum or salva sample was 1TCID50 respectively, in calm sample was 1-10 TCID50 . HAV RNA was detected in water and sera samples collected from the HAV outbreak region, sequenced and analysis. Conclusion The method developed here is convenient, specific and capable of detecting low levels of HAV in different samples, would be useful for diagnostic laboratories in order to perform HAV analysis in cases of foodborne infections or for molecular epidemiology investigation of HAV outbreaks.