基于单标记探针实时荧光定量PCR和熔解曲线分析方法定量检测HBeAg阳性患者前C区变异株

摘要

Objective To establish a proper systematic quantitative research approach which is used to detectcomplex hepatitis B virus precore mutants. Methods Accomplished the test via Real-time PCR in two steps. Firstly, the amplification of wild-type strains would be suppressed usingwild-type strain blocking probe (WT-blocker probe) to selectively inhibit the PCR (siPCR) whereas the mutant-type strains with low level could be amplified about 10 000 times, and be easily detected in the subsequent reaction; Secondly, precore mutants of G1896A, G1899A, G1896A/G1899A were quantitatively detected using single labeled probe combined with melting curve analysis system. Primer-blocker-probe partial-overlap (PBPPO) was designed to reduce the influence of gene polymorphism and to improve accuracy of detection. . Results would be confirmed by direct sequencing and analysis of polymerase chain reaction -restriction fragment length polymorphism (PCR-RFLP). Results Four standard plasmids (G1896/G1899, G1896A, G1899A, G1896A/G1899A) could be well detected and distinguished withmutants detection sensitivity reaching 0. 01% ; Among 10 HBeAg-positive patients with mutant negative identified by direct sequencing, at least one precore mutant was detected of each patient. Conclusion The single labeled probe fluorescent quantitative PCR combined with melting curve analysis system could be used for early sensitive detection of precore mutants Precore mutants evolving process and mechanism would be investigatedusing large-scale and longitudinal cohort study in future.%目的 建立一种HBV复杂前C区变异株精确定量的检测系统.方法 分两步实时PCR完成检测.首先应用野生株阻断探针(WT-blocker probe)进行选择性抑制野生株病毒的扩增,而含量偏低的变异株病毒得以扩增约10 000倍,使其在后续反应中易于检出;第二步应用单标记探针(SimpleProbe)结合熔解曲线分析系统精确定量检测前C区G1896A、G1899A、G1896A/G1899A变异株.通过PBPPO(primer-blocker-probe partial-overlap)设计减少基因多态性对检测的影响,来提高检测精确度.检验结果经直接测序及聚合酶链反应-限制性片段长度多态性分析方法予以证实.结果 4种标准质粒(G1896/G1899、G1896A、G1899A、G1896A/G1899A)能够很好地被检出并予以区分,突变检测灵敏度达到0.01%;10例HBeAg阳性(直接测序法鉴定前C区变异阴性)患者血清标本中,每例至少1种前C区变异株被检出.结论 单标记探针实时荧光定量PCR和熔解曲线分析能够早期灵敏检测前C区变异株,前C区变异株进化过程及机制还需进一步大样本、纵向队列研究予以阐明.