Establishment of universal detection method for virus genome in sera from patients with acute liver damage

本课题以急性期无甲、B、丙型肝炎病毒标志物的急性肝损害患者为研究对象，排除药物性、酒精性和自身免疫性肝炎。首先对这些患者血清中的HBV和HCV进行了套式聚合酶链反应（PCR）和套式逆转录（RT）-PCR，分别检测了HBV-DNA和HCV-RNA的主要S区和5 ′-非编码区。17例患者中有4例HCV基因扩增，诊断为急性丙型肝炎，而HBV基因均未扩增。并以急性甲型肝炎患者和急性B型肝炎患者作为对照，用上述PCR方法检测病毒基因组。结果表明，选择合适的采样时机和有效的核酸提取方法是十分重要的。其次，对急性非甲、B、C型肝炎（包括2例重型肝炎）患者血清中无常规HBV标志物的庚型肝炎和无症状B型肝炎进行了庚型肝炎病毒（HGV）RNA 5 '非编码区和解旋酶区巢式RT-PCR和HBV-DNA前C区巢式PCR检测。10例患者中有8例HBV基因扩增，无一例HGV基因扩增。建立急性肝损害患者血清中病毒基因组通用检测方法的原计划发生了变化，但目前已明确，HBV的沉默感染是急性非甲、B、C型肝炎患者的主要病因。

Patients with acute liver damage, who were excluded drug induced, alcholic and autoimmune hepatitis and without serum virus markers of hepatitis A,B and C in acute phase, were studied as subjects in this project. Firstly to deny the existence of hepatitis B virus (HBV) and hepatitis C virus (HCV) in the sera obtained from these patients, nested polymerase chain reaction (PCR) for HBV-DNA at major S region and nested reverse transcription (RT) -PCR for HCV-RNA at 5'-noncoding region were performed. HCV gene was amplified from 4 of the 17 patients and they were diagnosed as acute hepatitis C,however HBV gene was not ampified none of them. Detection of virus genome by these PCR methods was performed in patients with acute hepatitis A and B as controls. It was comfirmed that the timing of sampling and the selection of efficient method for extraction of nucleic acids were important. Secondly to examine the participation of hepatitis G and silent hepatitis B without ordinary HBV markers in the sera from selected patients with acute hepatitis non-A,B,C including 2 fulminant hepatitis, nested RT-PCR for hepatitis G virus (HGV) -RNA at 5'-noncoding region and helicase region and nested PCR for HBV-DNA at precore region were performed. HBV gene was amplified from 8 of the 10 patients, however HGV gene was not ampified none of them. The original plan for the establishment of universal detection method for virus genome in sera from patients with acute liver damage has changed, but it has became clear that the silent HBV infection is the main cause of patients with acute hepatitis non-A,B,C.