分枝杆菌分型三重PCR检测方法的建立及应用

Establishment and Application of Mycobacterium Typing Triple PCR Detection Method

本研究旨在建立一种快速鉴定分枝杆菌的三重PCR方法,并比较分析其在临床检测中的可靠性。根据已发表的结核分枝杆菌、牛分枝杆菌和非洲分枝杆菌rv3036c基因,结核分枝杆菌rv1970f基因(RD7)和牛分枝杆菌pncA基因的序列,改造并设计合成了3对特异性扩增引物,建立了一种能对分枝杆菌样品进行初步鉴定的三重PCR方法。结果显示该方法可针对rv3036c、rv1970f和pncA基因分别扩增出大小为500、125和249bp的目的片段,能特异性检测出结核分枝杆菌(500和125bp两条带)和牛分枝杆菌(500和249bp两条带),并可将结核分枝杆菌、牛分枝杆菌与其他分枝杆菌加以区分。本方法的检测灵敏度为50pg/μL模板基因组DNA。对86株抗酸染色阳性菌进行三重PCR鉴定,鉴定结果与细菌16SrDNA和ITS序列测定结果一致,检测准确度为100%,优于生长特征和生化试验鉴定。

This study was aimed to establish a triple PCR method to rapidly identify Mycobacteriumspecies,and evaluate its testing reliability.Three pairs of primer that were respectively specific to rv3036 c,rv1970fand pncAgenes of Mycobacterium were designed to establish a triple PCR for preliminary identification of Mycobacterium tuberculosis（M.tuberculosis）,Mycobacterium bovis（M.bovis）and other Mycobacteriumspp.PCR products were the expected sizes of 500（rv3036c）,125（rv1970f）and 249bp（pncA）,and contained two DNA bands（500and 125bp）with M.tuberculosis DNA template,two DNA bands（500and 249bp）with M.bovis DNA template.No band or non-specific band appeared with Mycobacteriumspp.except M.tuberculosis and M.bovis DNA templates.The sensitivity of the triple PCR was calculated to 50pg/μL template of genomic DNA.86acid-fast bacteria were detected by the triple PCR,16 SrDNA and ITS gene sequencing,growth test and biochemical test,and the results were consistent between triple PCR and 16 S rDNA and ITS gene sequencing.The detecting accuracy of triple PCR was 100%,and higher than growth test and biochemical test.