应用PCR技术快速鉴定结核分枝杆菌复合群菌种

Rapid species identification of Mycobacterium tuberculosis complex by PCR

目的建立快速鉴定结核分枝杆菌复合群菌种的方法。方法将7对结核分枝杆菌复合群各菌种特异性引物(M tbc1-7)进行聚合酶链反应(PCR)扩增,通过对扩增产物的不同系带型进行分析,对已知标准株和96株临床分离株进行菌种鉴定。结果用3株结核分枝杆菌复合群(MTBC)标准株,9株非分枝杆菌标准株以及27株非结核分枝杆菌的分枝杆菌(MOTT)对7对结核分枝杆菌复合群各菌种特异性引物进行特异性鉴定,结果证明引物M tbc1能鉴定出分枝杆菌和非分枝杆菌;引物M tbc2能鉴定出MOTT和MTBC;引物M tbc3能鉴定出田鼠分枝杆菌;引物M tbc4能从MTBC中识别出牛分枝杆菌和卡介苗;引物M tbc5能鉴定出非洲分枝杆菌Ⅱ型和结核分枝杆菌;引物M tbc6和引物M tbc4结合能鉴定出卡介苗;引物M tbc7和引物M tbc5联合能鉴定出canettii分枝杆菌。96株临床分离株经7对引物扩增,扩增结果显示MOTT 20株,牛分枝杆菌1株,卡介苗1株,canettii分枝杆菌3株,田鼠分枝杆菌3株,非洲分枝杆菌Ⅰ型3株,caprae分枝杆菌1株,结核分枝杆菌或非洲分枝杆菌Ⅱ型64株。结论应用PCR技术鉴定结核分枝杆菌复合群菌种结果准确可靠,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。

Objective To develop a new, rapid and easy method for species identification of Mycobacterium tuberculosis complex （MTBC）. Methods Seven sets of primers （Mtbcl-7） for MTBC were used to amplify the genomic DNA from the reference strains and 96 clinical isolates. Results The specificities of 7 sets of primers were identified by PCR with 3 MTBC reference strains, 9 nonycobacterium reference strains and 27 non-tuberculosis mycobacterium （NTM） reference strains. The results showed that mycobacterium and non-mycobacterium were identified by Mtbcl ; NTM and MTBC by Mtbc2 ; M. microti by Mtbc3 ; M. bovis and M. boris BCG by Mtbc4; M. africanum subtypeI and M. tuberculosis by Mtbc5; M. bovis BCG by Mtbc4 and Mtbc6 together; M. canettii by Mtbc5 and Mtbc7 together. Of 96 clinical isolates, 20 MOTT, 3 M. microti, 1 M. boris, 1 M. boris BCG, 3 M. africanum subtype Ⅰ , 1 M. caprae, 3 M. canettii, 64 M. africanum subtype Ⅱ and M. tuberculosis were identified. Conclusion PCR is a rapid and effective method for the species identification of MTBC, and can be used for molecular epidemiology.