【作者】 赵晨燕；

【导师】 王佑春；

【作者基本信息】 中国药品生物制品检定所 ， 病原生物学， 2006， 硕士

【摘要】 戊型肝炎病毒（HEV）是通过粪-口途径传播的无包膜、正链RNA病毒。它能引起大规模爆发性流行，也可引起急性散发性肝炎。目前戊肝的诊断主要是通过ELISA（酶联免疫法）检测抗HEV抗体，但因为HEV感染窗口期或患者自身免疫体制以及EIA试剂本身的原因等都会给戊肝的诊断带来难度；HEV RNA检测是判断HEV病毒是否存在的标准之一，但常规RT-PCR方法操作繁琐、容易污染、敏感性低，鉴于此本课题建立了一种快速、敏感、特异性强的实时荧光RT-PCR方法来检测戊型肝炎病毒。 本研究在建立实时荧光RT-PCR方法时，首先根据ORF3保守区域（5487—5613nt）设计了26条引物和4条探针，通过对引物和探针的筛选，确定了最佳的引物（401U／402R）和探针（301F）组合，并通过对引物、探针用量的优化确定40μl体系上下游引物用量分别为4pmol和8pmol，探针用量为8pmol；其次，通过对反应体系和条件进行优化，确定了Taq酶用量为4U、AMV逆转录酶的用量为2U、逆转录温度为50℃、PCR扩增采用三步法等；最后，通过对HEV RNA抽提试剂进行比较，选择使用QIAamp Viral RNA Mini Kit抽提HEV RNA。用建立的荧光PCR方法对9个不同浓度的假病毒稀释物进行了检测，结果显示，本套试剂的检测范围为5.6×10³～5.6×10¹⁰copies，最低可检测5.6×10³copies的假病毒分子，标准曲线的线性相关系数也较好，其为0.9968。 用该方法对149份临床急性肝炎患者血清进行检测，结果有65例为HEV更多还原

【Abstract】 Hepatitis E virus (HEV) is transmitted by the fecal-oral route and causes sporadic and epidemic hepatitis. So far, the HEV infection has been diagnosed mainly by detection of anti-HEV antibodies using ELISA method. However, the limitation of HEV antibody detection in window period of HEV infection, in patients with deficient immunity or even by using EIAs with different sensitivity and specificity may make the diagnosis difficult. Detection of HEV RNA is one of criteria for HEV infection. Conventional RT-PCR assays including nested RT-PCR have been used. However, the sensitivity is not enough and test procedure is time consuming. They are also prone to contamination when performed as nested RT-PCR procedures. To overcome the disadvantage of conventional RT-PCR assays, a rapid and sensitive real-time RT-PCR assay for the detection of HEV RNA was developed in this study.26 primers and 4 probes were designed based on a multiple sequence alignment of HEV ORF3 conserved region available in GenBank. By screening primers and probes, the best combination which includes forward primer (401U), the reverse primer (402R) and the probe (301F) had been determined. By optimizing the concentration of primer and probe, the concentration for forward primer, reverse primer and probe is .10pmol/μl, 0.20pmol/μl and 0.20pmol/μl, respectively. Then, the reactive parameters and condition including Taq DNA polymerase, AMV reverse transcriptase, the temperature of reverse transcription and PCR cycling had also been optimized. The concentration for Taq DNA polymerase and AMV reverse更多还原