实时荧光定量检测HCV RNA含量的临床意义  

Clinical significance of HCV RNA loads quantified by real-time fluorescence quantitative reverse transcription-PCR

作　　者：张冬雷[1] 施健[1] 崔之础[1] 王惠民[2] 鞠少卿[2] 李立人[3] 倪红兵[2] 苏建友[2] 

机构地区：[1]南通大学附属医院酶学研究室,江苏省南通市226001 [2]南通大学附属医院检验医学中心,江苏省南通市226001 [3]南通大学附属医院消化内科,江苏省南通市226001

出　　处：《世界华人消化杂志》2005年第1期39-43,共5页World Chinese Journal of Digestology

摘　　要：目的:建立实时荧光定量RT-PCR(RFQ-RT-PCR)检测HCV RNA含量的方法,初步探讨HCV RNA含量变化与病情的相关性. 方法:HCV抗体阳性样本80例,包括慢性肝炎(CH) 39例,肝硬化(LC)23例,肝癌(HCC)18例,献血员样本40例为正常对照.在HCV基因组5’非编码区(5’UTR)设计特异性引物和一对杂交探针,构建相应基因片段载体,体外转录RNA作为标准品,根据其建立的标准曲线对血清HCV RNA进行定量,同时用传统的套式RT-PCR进行定性分析. 结果:本法检测HCV RNA含量的线性范围为1012-104copies/L,批内和批间重复性测定的v分别为1.68-5.97%和6.40-10.58%.在HCV抗体阳性患者中, 肝硬化和肝癌患者的HCV RNA含量显著高于慢性肝炎患者(7.75±0.29,7.86±0.32 vs 4.67±0.41,P<0.05), 而且HCV RNA含量与ALT水平呈正相关(r=0.89, t=8.29,P<0.05). 结论:RFQ-RT-PCR检测HCV RNA含量具有较好的灵敏度和特异性,其含量与丙型肝炎患者的病情变化及严重程度有一定相关性.AIM: To establish real-time fluorescence quantitative reverse transcription- polymerase chain reaction (RFQ-RT-PCR) for quantification of HCV RNA, and to explore the relationship between HCV RNA loads and disease progress. METHODS: A total of 80 patients with anti-HCV positive were studied, including 39 with chronic hepatitis, 23 with liver cirrhosis and 18 with hepatocellular carcinoma. Forty samples from blood donors were used as normal controls. The specific primers and hybridization probes were designed in 5' untranslated region of HCV genome, and the corresponding gene fragment was cloned into pGEM-T vector to gain the RNA standards in vitro. According to the curve created based on RNA standards, serum HCV RNA was quantified by RFQ-RT-PCR, and was also qualified by nested RT-PCR simultaneously. RESULTS: The detected range of HCV RNA by RFQ-RT- PCR was from 1012 to 104copies/L, and the coefficient of variation values for both intra-experimental and inter-experimental reproducibility ranged from 1.68%, 6.40% to 5.97%, 10.58%, respectively. The mean loads of HCV RNA in liver cirrhosis and hepatocelluar carcinoma patients were significantly higher than those in chronic hepatitis patients (7.75±0.29, 7.86±0.32 vs 4.67±0.41, P<0.05), and there was also a positive correlation between HCV RNA loads and ALT level (r= 0.89,t= 8.29, P<0.05). CONCLUSION: RFQ-RT-PCR assay has good sensitivity and specificity in detection of HCV RNA loads, and HCV RNA loads may be associated with the progress and severity of disease state.

关 键 词：HCV抗体 RNA 含量 实时荧光定量 患者 阳性 慢性肝炎 RT—PCR 基因组 体外转录 

分 类 号：R446.5[医药卫生—诊断学]