El-Eragi A M S, Hamid M E, Saeed N S, Ahmed A H, Lee K, Kook Y-H, Mukhtar M M
Central Veterinary Research Laboratories, Soba, Khartoum, Sudan.
Int J Tuberc Lung Dis. 2007 Jun;11(6):671-5.
To characterise mycobacterial clinical isolates based on amplification of the rpoB gene.
One hundred and thirty-five mycobacterial isolates cultured from suspected pulmonary tuberculosis (TB) patients were identified phenotypically. Molecular characterisation of the isolates was performed based on amplification of the rpoB gene, using duplex polymerase chain reaction (DPCR), PCR-restriction fragment length polymorphism (RFLP) and nested PCR-based sequence analysis techniques.
The DPCR assay identified 129 of 135 (95.5%) clinical isolates as Mycobacterium tuberculosis complex species. Restriction enzyme analysis of the rpoB PCR product using Hind II identified 134 of the 135 (99.3%) isolates as M. tuberculosis complex, while nested PCR sequence analysis of the rpoB gene identified 133/133 examined isolates (100%) as M. tuberculosis species. No mycobacteria other than M. tuberculosis (MOTT) were detected among the studied isolates.
DPCR, PCR/RFLP Hind II and nested PCR sequence analysis of the rpoB gene techniques showed comparable efficiency in the characterisation of Mycobacterium isolates. Nested PCR sequence analysis of the rpoB gene was superior to PCR/RFLP for characterisation of suspected M. tuberculosis isolates, while the DPCR technique showed less sensitivity. As PCR-RFLP requires less sophisticated laboratory facilities than nested PCR sequence analysis, it would be more appropriate to be adopted for accurate characterisation of mycobacteria in countries with a weak infrastructure.
基于rpoB基因扩增对分枝杆菌临床分离株进行特征分析。
对从疑似肺结核患者中培养出的135株分枝杆菌分离株进行了表型鉴定。采用双重聚合酶链反应（DPCR）、聚合酶链反应-限制性片段长度多态性分析（PCR-RFLP）和基于巢式PCR的序列分析技术，基于rpoB基因扩增对分离株进行分子特征分析。
DPCR检测法将135株临床分离株中的129株（95.5%）鉴定为结核分枝杆菌复合群菌种。使用Hind II对rpoB PCR产物进行限制性酶切分析，在135株分离株中鉴定出134株（99.3%）为结核分枝杆菌复合群，而对rpoB基因进行巢式PCR序列分析，在133株检测的分离株中鉴定出133株（100%）为结核分枝杆菌菌种。在所研究的分离株中未检测到除结核分枝杆菌（非结核分枝杆菌）以外的分枝杆菌。
rpoB基因的DPCR、PCR/RFLP Hind II和巢式PCR序列分析技术在分枝杆菌分离株特征分析中显示出相当的效率。rpoB基因的巢式PCR序列分析在疑似结核分枝杆菌分离株的特征分析方面优于PCR/RFLP，而DPCR技术的敏感性较低。由于PCR-RFLP所需的实验室设施不如巢式PCR序列分析复杂，因此在基础设施薄弱的国家，采用PCR-RFLP进行分枝杆菌的准确特征分析更为合适。
