逆转录—聚合酶链反应在丁型肝炎病毒感染的应用及意义  

Detection of Hepatitis D Virus RNA by Reverse Transcription and Nested Polymerase Chain Reaction in Infected Serum and Liver Tissues

作　　者：黄德庄[1] 张建成[1] 闫惠平[1] 郎振为[1] 崔世昌[1] 贺丽香[1] 齐文杰[1] 

机构地区：[1]北京佑安医院肝炎研究所

出　　处：《中国医师杂志》1999年第6期11-13,15,共4页Journal of Chinese Physician

摘　　要：本项技术选取丁型肝炎病毒（ＨＤＶ）ＲＮＡ的非抗原编码区的相对保守区为靶序列，设计两对引物，进行巢式聚合酶链反应（ＰＣＲ）扩增ＨＤＶＲＮＡ。血清及肝组织采用蛋白酶消化法提取ＲＮＡ。结果发现９８例各型病毒性肝炎血清中，ＨＤＶ—Ｍ阳性组ＨＤＶＲＮＡ检出率为５６６％（３０／５３），ＨＤＶ—Ｍ阴性组为８９％（４／４５）（Ｐ＜００１）；ＨＤＡｇ或／及抗ＨＤＩｇＭ阳性血清测定ＨＤＶＲＮＡ的阳性率为５７１％（２８／４９），抗ＨＤ阳性血清的阳性率为５００％（２／４）；９例ＨＤＶ—Ｍ阳性肝组织测定ＨＤＶＲＮＡ阳性者６例，２例ＨＤＶ—Ｍ阴性肝组织均阴性。本项技术是在分子水平上检测病毒核酸、操作简便、稳定、可靠、特异性高、重复性好，可以作为ＨＤＶ血清学指标的佐证和补充，肝组织ＨＤＶＲＮＡ的测定可应用于回顾性研究，在临床病原诊断、抗病毒疗效判定、病毒复制、重叠感染等研究方面提供重要实验依据。We established a method of reverse transcription and nested polymerase chain reaction (RT-PCR) for the detection of hepatitis D virus (HDV) RNA.Two pairs of primers were designed which were located in the conserved sequences of non-antigen coding region of HDV genome.The inner primers were 366bp nucleotides in length.RNA was extracted by proteinase K digestion method from human infected serum or paraffin-embedded liver tissues.The results showed that in the whole of 98 hepatitis cases sera,the detective rate of HDV RNA by RT-PCR in HDV-M positive group was 56 6%(30/53),and in HDV-M negative group was 8 9%(4/45) (comparison of two groups: P <0 01).In HDAg and /or IgM antiHD positve serum,the positive rate of HDV RNA was 57 1%(28/49),and in total antiHD positive serum was 50 0%(2/4)。For the infected liver tissues detection:from 9 of HDV-M positive cases liver tissues,HDV-RNA were detectable in 6 cases; and from 2 of HDV-M negative cases liver tissues,HDV RNA were negative.HDV RNA From 11 cases liver tissues were studied by using in situ hybridization (NISH) assay with a digoxingenin-labelled probe.The results of NISH and RT-PCR for the detection of HDV RNA were compared,the corresponsable rate was 81 1%(9/11,χ 2=0 5, P >0 05).Our study suggest that the RT-PCR for HDV RNA was stable highly sensitive and specific,and that was an useful technique for the study on clinical and experimental field in hepatitis D virus infection.

关 键 词：丁型肝炎病毒 逆转录 PCR 

分 类 号：R512.63[医药卫生—内科学]