应用PCR-SSCP银染技术检测HBV基因点突变

Detecting the Mutation of HBV by Using PCR-SSCP and Silver Staining Technique

为了对乙型肝炎患者HBV基因突变进行大样本大面积筛检,建立简便可靠的检测HBV基因突变的方法。方法:采用单链构象多态性(SSCP)银染技术,检测HBV Pre-C基因突变。结果:在10份HBeAg阳性标本中,有8份PCR阳性,SSCP显示有2份标本有HBV Pre-C基因突变,直接测序证实为非终止密码突变。48份抗-HBe阳性标本中,有12份为PCR阳性,有8份SSCP显示PCR产物单链泳运状态异常,其中4份标本经直接测序证实在1898位发生了G→A变异,出现TAG终止密码突变。结论:PCR-SSCP银染技术检测HBV Pre-C基因点突变有很高的特异性和敏感性,该方法稳定、简便、快速,能基本满足临床大面积大样本筛检的需要。

To establish a convenient and reliable detecting method for prescriptive screening the mutation of gene of HBV on a larg scale. Methods:The single strand conformation polymorphism analysis of polymerase chain reaction (PCR-SSCP) products and silver staining technique were used for detecting the precore mutation of hepatitis B virus. Results: In the experiment, among ten positive serum samples of HBeAg, eight were seropositive for HBV DNA by detection uising PCR. and then the two of them exhibited mutation of Pre-C gene of HBV by further detection using SSCP technique. The fact that the mutation for these two samples were in character of non stopping code was demonstrated by direct detecting the range; among forty-eight positive samples of anti-HBe, twelve were seropositive for HBV DNA by detection using PCR, and then the PCR products were further detected by using SSCP technique and found that the single chain electrophoresis of eight samples were in a unusual state. The eight sample were funther studied by direct detecting the ronge and the results showed that the four of then had produced the mutation of TAG stop code because of a change from G to A at the point of 1898. Conclusidns: PCR-SSCP and silver staining technique is very peculiarity and sensitively to detecting the mutation of Pre-C gene of HBV. The method is very stable,simple and fast, which can satisfy the needs of prescriptive screening of samples on a larg scale.