RTQ-PCR检测肺炎支原体2063位点及其突变基因的应用

Application of real-Time PCR for determining gene mutation of 2063 point in Mycoplasma Pneumoniae

目的建立一种快速诊断肺炎支原体(MP)感染及其耐药表型的新方法。方法采用实时荧光定量PCR(RTQ-PCR)技术,以23S rRNA为靶序列,设计药物敏感2063A质粒和耐药2063G质粒的基因型探针及引物,构建药物敏感2063A质粒和耐药2063G质粒的RTQ-PCR定量标准曲线,并对他们进行相关性分析;对100份临床咽拭子标本进行2063A质粒和耐药2063G质粒RTQ-PCR检测,分析其检测结果。结果 RTQ-PCR检测药物敏感2063A质粒和耐药2063G质粒与23S rRNA巢式PCR检测序列结果比较,RTQ-PCR鉴定100株MP临床分离株2063位点等位基因的灵敏度、特异性、阳性预测值、阴性预测值均为100%。100份临床咽拭子标本MP阳性检测率为83%(83例),耐药2063G质粒33%(33例),8%(8例)同时发现药物敏感2063A质粒和耐药2063G质粒。结论本研究初步建立了一种快速诊断MP感染和其耐药基因型的RTQ-PCR方法,具有较高的灵敏度与特异性,可以在临床上推广。

Objective To set up a new real time approach for determination of 2603 point mutation drug resistant gene of Mycoplasma pneumoniae（MP）.Methods The allelic discrimination of 2063 point mutation of MP was detected by real-time PCR（RTQ- PCR） with 23S rRNA of MP as the target.Results Compared with the results of 23S rRNA netsted PCR sequencing,the sensitivity,specificity,positive value,negative predictive value of RTQ-PCR identifying 2063 allele of 100 MP isolates were all 100%,MP positive detection rate,drug resistance rate of 2063G plasmid of 100 nasopharyngeal swabs were83%（83 cases）,33%（33 cases）,respectively.Loci 2063A,2063G were simultaneously detected in 8%（8 cases）.Conclusion Preliminarily a RTQ-PCR method for fast diagnosis of MP infection and judgment its resistance genotypes was established with high sensitivity and suitable for clinical application.