多位点PCR用于安徽391株分枝杆菌临床分离株的快速鉴定

Rapid species identification of 391 clinicalMycobacterium isolates from Anhui Province by multi-locus PCR

目的评价多位点PCR用于分枝杆菌临床分离株快速鉴定效果。方法收集从临床结核病患者分离到的抗酸染色阳性的培养物,经PNB/TCH鉴别培养基进行培养鉴定后,采用聚合酶链反应(PCR)对16SrRNA、Rv0577、IS1561、Rv1510、Rv1970、Rv3877/8和Rv3120基因位点进行扩增,鉴定至种,再经rpoB-PRA、hsp65和rpoB基因测序进行验证。结果共对391株分枝杆菌临床分离株应用多位点PCR进行了鉴定,结果显示结核分枝杆菌378株,非洲分枝杆菌I型6株,非结核分枝杆菌7株。7株非结核分枝杆菌分别为鸟分枝杆菌1株,马赛分枝杆菌2株,胞内分枝杆菌4株。而PNB/TCH鉴别培养基培养鉴定结果为结核分枝杆菌复合群385株,非结核分枝杆菌6株。多位点PCR结果与rpoB-PRA、hsp65和rpoB基因测序结果一致。结论多位点PCR技术鉴定分枝杆菌菌种结果准确可靠,且具有简便和快速等优点,有较大的分子流行病学应用价值,且对于临床诊断和治疗都具有重要意义。

The clinical Mycobacterium isolates were collected for the research to evaluate effect for rapid species identification. Primarily species were identified by culture method with differentiated PNB/TCH media. By means of multi-locus poly- merasechain reaction （multi-locus PCR）, the specific DNA fragments of 16SrRNA, Rv0577, IS1561, Rvl510, Rv1970, Rv3877/8 and Rv3120 genes in the genome of the isolates were examined respectively. The species were confirmed by rpoB- PRA and sequencing of bsp65 and rpoB. A total of 391 Mycobaeterium isolates were collected for the species identification. By multi-locus PCR, 378 were identified as Mycobacterium tuberculosis, 6 were Mycobacterium africanum I type, and 7 were nontuberculous mycobacteria （NTM）. The 7 NTM strains were confirmed to be one Mycobacterium avium, 2 Mycobacterium massiliense, and 4 Mycobacterium intracellular. The results of multi-locus PCR were the same as that of rpoB PRA and se quencing of hsp65 and rpoB. While by culture method with differentiated PNB/TCH media, 385 strains were identified as My- cobacterium tuberculosis complex and 6 was NTM. Therefore, it＇s concluded that multi locus PCR in this study could identify Mycobacterium species accurately, reliably, simply and rapidly. It has significantly useful value for the molecular epidemiolo- gy, clinical diagnosis and clinical therapy of Mycobacterium tuberculosis.