一种竞争性RT-PCR测定HCV RNA方法的建立及其应用  

Development and Application of a Quantitative Assay by Competitive RT-PCR to Measure Serum HCV RNA

作　　者：杨才生 卢桥生[2] 李东良 温云海 张国安 

机构地区：[1]解放军476医院,福建福州350002 [2]第一军医大学南方医院,广东广州510515

出　　处：《现代诊断与治疗》2000年第3期155-157,共3页Modern Diagnosis and Treatment

摘　　要：目的　建立检测丙型肝炎病人血清HCVRNA水平的竞争性逆转录 聚合酶链式反应定量方法。方法　采用含HCV 5 非编码区 ( 5 NCR)缺失突变的重组质粒 5T1d ,将其目的基因亚克隆到体外转录载体pCDNAⅠ多克隆位点上 ,获得转录重组体 5T1d 。将其线性化后用SP6RNA聚合酶体外转录竞争性缺失突变模板RNA ,该模板定量后与血清HCVRNA一起作逆转录 聚合酶链式反应 (RT PCR) ,建立检测血清HCVRNA水平的竞争性定量方法。结果　检测 15例丙肝病人 2 0份血清 ,滴度为 6.6× 10 2 ～ 6.6× 10 6copies/ml。 结论　各类丙型肝炎患者血清病毒滴度均较高 ,经干扰素治疗后病毒水平下降 1～ 5个Log ,年龄小于 2 0岁者干扰素治疗效果较好。Objective To establish quantitative assay by competitive RT PCR measuring serum HCV RNA from patients with HCV infection.Method An in vitro transcription recombinant plasmid 5T1d was obtained after subcloning the target gene of clone 5T1d,which contained mutant HCV 5 non coding region cDNA,into a transcriptive vector pCDNA Ⅰ.After linearization of 5T1d,recombinant RNA was synthesized using SP6 RNA polymerase.A competitive RT PCR assay was established by co amplification HCV wild type genome extracted from serum and recombinant RNA.Results The HCV RNA titers of 20 sera from 15 cases with hepatitis C,including five patients treated with alpha interferon(IFN α),were detected using this method.It was found that serum HCV RNA concerntrations ranged from 6.6×10 2 to 6.6×10 6 copies/ml.Conclusions There were high titers of HCV RNA in all kinds of patients with HCV infection.In the five patients treated with IFN α,the serum concentrations decreased 1～5Log.The effect of IFN α is better in patients younger than 20 years old.

关 键 词：丙型肝炎病毒 逆转录-聚合酶链反应 HCV-RNA 

分 类 号：R512.630.4[医药卫生—内科学]