非结核分枝杆菌耐药性特征及聚合酶链式反应检测方法的建立

The development of PCR assay for the rapid detection of non-tuberculous mycobacteria

目的建立一种快速鉴别非结核分枝杆菌的聚合酶链式反应方法，并与直接测序法比较，探讨该方法的可行性。方法根据分枝杆菌16—23srDNA间隔区序列设计引物，建立检测分枝杆菌的PCR分型方法，并利用该技术对5例临床分离株进行PCR扩增。借助凝胶电泳分析和直接测序分析进行临床分离株的分子分型。结果5例分离株均耐利福平、异烟肼、乙胺丁醇、链霉素、丁胺卡那、环丙沙星、左氧氟沙星、硫异烟胺、力克肺座和比嗪酰胺，为多重耐药菌株。PCR扩增结果和直接测序结果相吻合，均为脓肿分枝杆菌。结论分枝杆菌高度保守的16～23srDNA间隔区序列可做为分枝杆菌鉴定的靶基因，建立快速、特异、敏感的PCR扩增体系，且从基因水平上可对分枝杆菌进行分子分型．为临床治疗提供快速、准确的实验数据。

Objective To develop a simple and rapid identification system for non-tuberculous mycobacteria, comparing with PCR direct sequencing and drug sensitivity test to evaluate the method. Methods Based on the sequences of 16- 23srDNA intergenie spacer gene published on GenBank, a set of primers was designed, and the PCR assay for the rapid detection of non-tuberculous myeobacteria was developed. DNA of 5 clinical samples was amplified separately, and then electrophoresis and direct sequencing were used for sub-typing. Results 5 clinical strains were all resistant to RFP, INH, EB, SM, BB-K8, CIP, LOFX, TH3624, DPC and PZA.PCR amplification result was in agreement with direct sequencing result. 5 clinical samples were all Mecobanterium abscessus. Conclusions 16-23srDNA sequence can be used as target genes in mycobacterial identification. The developed PCR assay has the advantage of rapidity, sensitivity and specificity. It could be applied to the rapid detection of non-tuberculous mycobacteria and provide accurate testing data for clinical treatment.