探针ASPCR检测肺炎支原体微量耐药突变A2063G方法的建立

Development of probe-based Allele-specific real-time PCR testingfor detection of minor A2063G resistance mutation in Mycoplasma Pneumoniae

目的 建立能够特异性检测微量肺炎支原体(Mycoplasma pneumoniae, MP)A2063G耐药突变基因的特异性扩增等位基因的探针法实时定量PCR(probe-based allele-specific real-time PCR,探针ASPCR)方法。方法 建立特异性检测A2063G耐药突变位点的探针ASPCR方法,并验证其灵敏度、特异度及准确度等性能。结果 特异性扩增2063G和非特异性扩增2063A/G的引物/探针组合分别扩增105拷贝野生基因型(2063A)模板的Ct值的差(△Ct)高达10.93,能够特异性检测A2063G突变。探针ASPCR方法检测2063G基因型占总MP的比例的准确度可低至1%;检测MP的灵敏度低至10拷贝,检测A2063G耐药突变比例的灵敏度低至0.01%。探针ASPCR方法与前期建立的染料ASPCR方法检测临床样本的MP感染结果一致,MP阳性检出率均为94.83%(55/58),高于传统巣式PCR联合测序方法的检测结果(75.86%,44/58);染料ASPCR方法检测MP耐药率为70.91%(39/55),高于探针ASCR方法的检测结果 63.64%(35/55)。结论新建探针ASPCR方法是一种具有高特异度、准确度和灵敏度的快速检测MP微量A2063G耐药突变的方法;与染料ASPCR方法相比,探针ASPCR方法检测耐药MP的灵敏度略低,但其临床样本检测复查率也低于染料ASPCR方法,且其结果判读简单,更适合在临床中应用推广,能够为临床制定MP及耐药MP感染的治疗方案提供理论依据。

Objective Develope a probe-basedallele-specific real-time PCR to detect and analyze the A2063G resistance mutation of Mycoplasma pneumoniae(MP). Methods The ASPCR method using probe was developed to detect A2063G drug resistance mutation, and the specificity, sensitivity and accuracy of the method wereverified. Results The difference of Ct value( △ Ct) between specific and non-specific primer/probe combinations amplification of 105 copies of wild-type template was 10.93.The sensitivity of MP detection was down to 10 copies, and the sensitivity of mixed drug resistance mutation detection was down to 0.01%. The accuracy of detecting the mutation proportion was as low as 1%. The results of MP infection detected by the probe ASPCR method were consistent with those of the dye ASPCR established earlier, and the positive detection rates of MP were all 94.83%(55/58), higher than the traditional nested PCR detection results(75.86%, 44/58). Drug-resistant MP detection results: the resistance rate of MP detected by nested PCR combined sequencing was 59.09%(26/44);the drug resistance rate of MP detected by probe ASPCR and dye ASPCR was 63.64%(35/55) and 70.91%(39/55) respectively. Conclusions The probe ASPCR we developed is a rapid, sensitive and accurate method for detecting MP minor resistance mutations. In clinical samples,Tthe sensitivity of probe ASPCR to detect drug-resistant MP was slightly lower than that of dye-based ASPCR, but its clinical sample detection review rate was lower than that of dye-based ASPCR, and its results were easy to interpret, so it was more suitable for clinical application and popularization. And the probe ASPCR we developed can provide a theoretical basis for clinical development of MP and drug-resistant MP infection treatment plan.