丙型肝炎病毒核酸的荧光定量检测及其临床意义

目的:建立实时荧光定量RT-PCR(RFQ-RT-PCR)检测HCV-RNA含量方法的临床应用价值。方法: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,PCR扩增目的片段并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,同时用传统的巢式RT-PCR进行定性分析。结果:138例样本在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(P<0.05),而且HCV RNA含量与ALT水平呈正相关(r=0.91)。结论:RFQ-RT-PCR检测HCV-RNA含量比ELISA法具有更好的灵敏度和特异性,能准确反映HCV在体内的复制情况。

Clinical significance of detection of HCV-RNA quantity by real-time fluorescence quantitative RT-PCR

Objective:to develop a new method for detection of HCV-RNA quantity by real-time fluorescence quantitative RT-PCR (RFQ-RT-PCR), and to approach its clinical value in diagnosis, treatment, and prevention of HCV infection. Methods:specific primers and probes have been designed in the highly conserved region of HCV 5' un-translation region (5'-UTR) , and fluorescence of the PCR product was detected continuously during amplification. According to the standard curve created by RNA standard, the load of HCV RNA in clinical samples has been determined by using software. Results:Serum samples from 138 hepatitis patients were tested by RFQ-RT-PCR and ELISA simultaneously. The results showed that the HCV RNA mean loads of liver cirrhosis and hepatocelluar carcinoma patients with anti-HCV antibody positive were significantly higher than those of chronic hepatitis patients(P<0.05), and there was also a positive correlation between HCV RNA loads and ALT level(r=0.91).Conclusions:the results showed RFQ-RT-PCR was more sensitive and specific than ELISA, and it can be used as a good monitor for the state of HCV infection and its complication.