ASPCR检测微量HIV-1 K103N耐药突变方法的建立

摘要

Objective To develop and evaluate the allele-specific real-time PCR(ASPCR) assay for the detection of minor HIV-1 variants. Methods We developed and evaluated the ASPCR assay, using the K103N mutation site as a model system. We constructed plasmids as standards and designed specific and non-specific primers to discriminate the wild-type and mutant plasmids in the real-time PCR using SYBR green as fluorescence reporter. And then we evaluated the sensitivity, accuracy, reproducibility of ASPCR assay and detected the control samples. Results The specific primer can discriminate the wild-type and mutant plasmids including resistant mutation successfully. The sensitivity of ASPCR assay can achieve less than 0.01% and the accuracy of this method is down to 0.1%. The Intra-assay coefficient of variation is less than 0.7 and the Inter-assay coefficient of variation is less than 1.6. Conclusion ASPCR is a sensitive, accurate and rapid method to detect the minor HIV-1 variants which have resistant mutations and it can be used widely in HIV research. ASPCR also can provide earlier and more resistant information to the clinical therapy.%目的 建立检测微量HIV-1 K103N耐药突变的等位基因特异扩增实时定量PCR(allele-specific real-time PCR,ASPCR)方法,用于微量K103N耐药突变的检测和分析.方法 首先建立检测K103N耐药突变的ASPCR方法,然后对方法进行验证,最后采用ASPCR方法检测对照样本.构建含K103N突变的质粒作为标准品,然后设计特异性引物用以区分野生型和突变型模板,采用SYBR green法进行实时定量PCR,并绘制标准曲线.分别采用特异性和非特异性引物扩增模板,根据二者Ct(cycle threshold)值结合相应的标准曲线判断是否存在K103N突变及突变比例.对ASPCR检测K103N突变位点的特异性、敏感性、准确性、重复性等进行验证.结果 特异性和非特异性引物扩增等量野生型模板的Ct值之差(△Ct)可达13.56;当突变比例小于0.1%时仍具有良好的准确性;批内变异系数小于0.7,批间变异系数小于1.6;检测灵敏度可达0.01%.检测阴性对照样本的△Ct显著高于临界值,阳性对照样本检测结果均为刚性.结论 ASPCR是一种快速、灵敏、准确的检测HIV-1微量耐药突变的方法,可为临床抗病毒治疗提供理论指导.