论文部分内容阅读
采用反转录聚合酶链反应（ＲＴＰＣＲ）结合ＤＮＡ杂交技术，直接检测急性戊型肝炎病人和实验感染猴粪便标本中的戊型肝炎病毒（ＨＥＶ）基因。将从粪便标本中提取的ＲＮＡ反转录合成ｃＤＮＡ后，用ＨＥＶ特异引物进行套式ＰＣＲ扩增，并用长臂光敏生物素标记的ＨＥＶＥＴ１．１核酸探针进行杂交。结果显示，直接从６份病人１０％粪悬液标本中提取的ＲＮＡ进行ＲＴＰＣＲ，在琼脂糖凝胶电泳上未见任何信号带，点杂交也未见阳性。但标本悬液再用ＰＥＧ沉淀后提取的ＲＮＡ，有３份标本检出单一带（约２４０ｂｐ），Ｓｏｕｔｈｅｒｎ杂交亦显示阳性。然后检测用ＨＥＶ８７Ａ株感染的恒河猴粪便标本，也呈阳性结果。这为ＨＥＶ的基因诊断提供了可靠而有效的途径。
Reverse transcriptase-polymerase chain reaction (RT-PCR) and DNA hybridization were used to directly detect hepatitis E virus (HEV) gene in acute hepatitis E patients and experimentally infected monkeys. The RNA extracted from the stool specimens was reverse transcribed into cDNA, then subjected to nested PCR with HEV-specific primers and hybridized with a long-arm biotin-labeled HEVET 1.1 nucleic acid probe. The results showed that RT-PCR was performed directly on RNA extracted from 10% of fecal samples from 6 patients, no signal band was observed on agarose gel electrophoresis, and no dot blot was found. However, RNA extracted from the sample suspension and then precipitated with PEG showed a single band (about 240 bp) in 3 specimens and a positive result by Southern blot. Rhesus monkey stool specimens infected with HEV87A strain were then tested for positive results. This provides a reliable and effective way for gene diagnosis of HEV.