两种多位点聚合酶链反应技术在结核分枝杆菌复合群鉴定中的价值

The value of rapid species identification of Mycobacterium tuberculosis by two kinds of multi-locus polymerase chain reaction in children

目的评价多位点聚合酶链反应(PCR)在儿童结核分枝杆菌复合群鉴定中的临床价值。方法收集经初步鉴定为结核病分支杆菌的临床分离株,分别经传统PNB/TCH鉴定,以及选择7基因位点16SrRNA、Rv0577、IS1561、Rv1510、Rv1970、Rv3877/8及Rv3120,及4基因位点ropB、RD1、RD8(present)、RD8(deleted),采用聚合酶链反应(PCR)对其进行扩增和鉴定。结果共收集204株临床分离菌株,传统PNB/TCH鉴定结果为,结核分枝杆菌199株,牛结核分枝杆菌3株,非结核分枝杆菌2株。4位点PCR鉴定结果为,结核分枝杆菌196株,牛结核分枝杆菌2株,卡介苗3株,非结核分枝杆菌3株。7位点PCR鉴定结果为,结核分枝杆菌191株,牛结核分枝杆菌2株,卡介苗3株,非洲分枝杆菌Ⅰ型4株,山羊分枝杆菌和田鼠分枝杆菌各1株,非结核分枝杆菌2株。结论两种PCR方法均较传统方法简便快捷,且均可较快的鉴定结核分枝杆菌复合群更多的亚种。7位点能鉴定出除非洲分枝杆菌Ⅱ型以外的所有儿童结核分枝杆菌复合群的亚种。4位点在鉴定卡介苗菌株中更加迅速简便。

Objective To evaluate the clinical value of multi-locus polymerase chain reaction （PCR） for identifying Mycobacterium tuberculosis complex isolated in children. Methods The isolates were collected and were first determined by PNB/TCH medium. 7-point PCR sites including 16SrRNA, Rv0577, IS1561, Rv1510, Rv1970, Rv3877/8 and Rv3120, and 4-point PCR sites including ropB, RD1, RD8 （present）, RD8 （deleted） were used to amplify them by PCR. Results Total of 204 isolates were collected, in which 199 were Mycobacterium tuberculosis, 3 were Mycobacterium bovis, and 2 were non-tuberculous mycobacteria by the PNB/TCH method. 4-point PCR analysis showed that 196 were Mycobacterium tuberculosis, 2 were Mycobacterium bovis, 3 were BCG species and 3 were non-tuberculous mycobacteria. 7-point PCR analysis showed that 191 were Mycobacterium tuberculosis, 2 were Mycobacterium bovis, 3 were BCG species, 4 were African Mycobacterium type I, 1 was Mycobacterium caprae, 1 was Mycobacterium microti and 2 were non-tuberculous mycobacteria. Conclusion Compared with the conventional method, the PCR identification in 4-point PCR method and 7-point PCR method could rapidly identify the BCG among the complex group in children tuberculosis. 7-point PCR method was able to identify all the subspecies of Mycobacterium, except Africa Mycobacterium. 4-point PCR method would be more rapid and easier in the identification of BCG strains.