摘要
目的研究和评估PCR扩增阻断联合荧光探针检测乙型肝炎病毒(HBV)基因前C区1896位点突变的新方法。方法根据HBVDNA前C区1896位点的突变设计一系列引物,引物3′末端位于1896位,并与突变模板1896位碱基互补.在引物的倒数第2及第3位碱基设计为错配碱基,以增加反应的特异性。结果以2个错配碱基的突变型引物对野生型质粒进行PCR扩增,具有显著的阻断作用,对1896位突变型质粒无阻断作用,而且对其检测的最低拷贝数可达5×103拷贝/ml。选用该引物对95例随机采集的HBV阳性标本进行HBV前C区G1896A位碱基突变的检测,8例为阳性,其突变率为8.4%。结论本法在临床标本检测中是一种有效的和简便的方法。
Objective To investigate and evaluate the method of PCR amplification blocking associated with fluorescent probe for the detection of pre-C region of HBV G1896A gene mutation. Methods The primers were designed based on the mutation of HBV DNA 1896 locus. The 3′ end of primers was at 1896 site, and it was complemented with the base sequence of mutation template of 1896 site. The mismatching bases were separately introduced into the second and the third base of the primer by inverse counting from the 3 ＇end for in- creasing the specificity of reaction. Results The PCR amplification for wild plasmid with the mutant primer showed an effectively blocking, but not showed blocking for the mutant plasmid （G1896A）. The sensitivity of detection for the mutant plasmid was 5 × 10^3 copies/ ml. Ninety-five cases of HBV-positive serum was selected randomly and amplified with the mutant primer, and 8 cases were positive HBV G1896A gene mutant（ mutant rate of 8.4% ）. Conclusion The amplification blocking associated with fluorescent probe for the detection of HBV G1896A gene mutation is a effective, convenient method for the detection of clinical samples.
出处
《临床检验杂志》
CAS
CSCD
北大核心
2007年第3期182-184,共3页
Chinese Journal of Clinical Laboratory Science
