Abass N A, Suleiman K M, El Jalii I M
Department of Preventive Medicine, College of Veterinary Medicine and Animal Production, Sudan University of Science and Technology, PO Box 204, Khartoum North, Sudan.
Indian J Med Microbiol. 2010 Jan-Mar;28(1):26-9. doi: 10.4103/0255-0857.58724.
To evaluate the reliability of the gyrB PCR-RFLP technique in differentiating clinical Mycobacterium tuberculosis complex isolates.
A primer pair MTUB-f and MTUB-r for M. tuberculosis complex (MTBC) was used to differentiate 79 mycobacterial isolates by specific amplification of the 1,020-bp fragment of the gyrB gene (gyrB-PCR1). The MTBC isolates were further differentiated using a set of specific primers MTUB-756-Gf and MTUB-1450-Cr that allowed selective amplification of the gyrB fragment specific for M. tuberculosis (gyrB-PCR2). The DNA polymorphisms in the 1,020-bp gyrB fragment for 7 M. tuberculosis strains confirmed by PCR as well as 2 reference strains; M. tuberculosis H37Rv and M. bovis BCG were analyzed with the restriction enzyme Rsa1.
Seventy-seven (97.5%) isolates were positive for gyrB-PCR1 and thus identified as members of M. tuberculosis complex (MTBC) and two (2.6%) isolates were negative and identified as Mycobacteria other than tuberculosis (MOTT). All the M. tuberculosis isolates showed the typical M. tuberculosis specific Rsa1 RFLP patterns (100, 360, 560-bp) while 360 and 480-bp fragments were generated from M. bovis BCG.
The gyrB PCR-RFLP using the endonuclease Rsa1 can be used to differentiate M. tuberculosis from M. bovis in clinical isolates.
评估gyrB PCR-RFLP技术在鉴别临床结核分枝杆菌复合群分离株方面的可靠性。
使用一对用于结核分枝杆菌复合群（MTBC）的引物MTUB-f和MTUB-r，通过特异性扩增gyrB基因的1020 bp片段（gyrB-PCR1）来鉴别79株分枝杆菌分离株。使用一组特异性引物MTUB-756-Gf和MTUB-1450-Cr进一步鉴别MTBC分离株，这组引物可选择性扩增结核分枝杆菌特异性的gyrB片段（gyrB-PCR2）。对7株经PCR确认的结核分枝杆菌菌株以及2株参考菌株；结核分枝杆菌H37Rv和牛分枝杆菌卡介苗的1020 bp gyrB片段中的DNA多态性，用限制性内切酶Rsa1进行分析。
77株（97.5%）分离株gyrB-PCR1呈阳性，因此被鉴定为结核分枝杆菌复合群（MTBC）成员，2株（2.6%）分离株呈阴性，被鉴定为非结核分枝杆菌（MOTT）。所有结核分枝杆菌分离株均显示出典型的结核分枝杆菌特异性Rsa1 RFLP模式（100、360、560 bp），而牛分枝杆菌卡介苗产生了360和480 bp片段。
使用内切酶Rsa1的gyrB PCR-RFLP可用于鉴别临床分离株中的结核分枝杆菌和牛分枝杆菌。
