建立基于gyrB基因检测结核分枝杆菌复合物的荧光定量PCR法

Establishment of fluorescence quantitative PCR for detection of Mycobacterium tuberculosis complex based on gyrB gene amplification

目的建立基于gyrB基因扩增检测结核分枝杆菌复合物(MTC)的荧光定量PCR(FQ-PCR)法。方法根据gyrB基因设计合成引物和探针,建立并评价TaqMan探针FQ-PCR法,并与基于IS6110序列的FQ-PCR法分别检测50例临床标本,比较两种方法的检测结果。结果建立的FQ-PCR法标准曲线方程为Y=-3.18X+42.84,相关系数(r2)为0.995、扩增效率为106.25%,其检测灵敏度为102CFU/mL;MTC中结核分枝杆菌和非洲分枝杆菌阳性,其他分枝杆菌和4株临床其他细菌均为阴性。批内及批间变异系数(CV)均<2%,重复性良好。本法与基于IS6110序列的FQ-PCR法对50例临床标本的检测结果差异无统计学意义(χ2=0.06,P>0.05),一致性好(κ=0.94)。结论基于gyrB基因扩增的FQ-PCR法灵敏度高、特异性强、重复性好,可用于结核分枝杆菌复合物的早期快速诊断。

Objective： To establish a fluorescence quantitative PCR （FQ-PCR） assay for the detection of Mycobacterium tuberculosis complex （MTC） by amplifying gyrB gene. Methods：The specific primers and TaqMan probe targeting gyrB gene were designed to establish FQ-PCR assay and the methodology of the assay was evaluated. Fifty clinical specimens were detected by the established FQ-PCR and another FQ-PCR based on amplification of IS6110 sequence, and the results of both assays were compared. Results：For the established FQ-PCR assay the standard curve equation was Y=-3.18X＋42.84, the correlation coefficient （r2） was 0.995, the amplification efficiency was 106.25%, and the lowest detectable limit was 102 CFU/mL. Mycobacteria tuberculosis and Mycobacterium africanum could be detected in MTC by the FQ-PCR assay, while other mycobacterium and four strains of other clinical bacteria were negative. Both the intra- and inter-assay coefficients of variation were less than 2% with fine reproducibility. No significant difference of the results between the two FQ PCR assays was found for 50 clinical specimens （χ2=0.06, P〉0.05）, and the results of both assays were remarkably consistent （κ=0.94）. Conclusion： The established FQ-PCR assay based on amplification of gyrB gene is sensitive, specific and reliable for the early and rapid diagnosis of Mycobacterium tuberculosis complex infection.