Donoghue H D, Holton J, Spigelman M
Department of Medical Microbiology, University College London, London W1T 4JF.
J Med Microbiol. 2001 Feb;50(2):177-182. doi: 10.1099/0022-1317-50-2-177.
There are several specific PCR-based methods to detect Mycobacterium leprae DNA, but the amplicons are quite large. For example, primers that target the 36-kDa antigen gene and are in common diagnostic use yield a 530-bp product. This may be a disadvantage when examining samples in which the DNA is likely to be damaged and fragmented. Therefore, two sets of M. leprae-specific nested primers were designed, based on existing primer pairs which have been shown to be specific for M. leprae. Primers that targeted the 18-kDa antigen gene gave an outer product of 136 bp and inner product of 110 bp. The primers based on the RLEP repetitive sequence yielded a 129-bp outer product and 99-bp nested product. With dilutions of a standard M. leprae killed whole-cell preparation as the source of DNA, both single-stage and nested PCR were performed after optimisation of the experimental conditions. Compared with the 36-kDa antigen gene primers, the 18-kDa antigen gene outer primers were 100-fold more sensitive and the RLEP outer primers were 1000-fold more sensitive. As an illustration of two possible applications of these new primers, positive results were obtained from three skin slit samples from treated lepromatous leprosy patients and three archaeological samples from human remains showing typical leprosy palaeopathology. It was concluded that these new primers are a useful means of detecting M. leprae DNA which is damaged or present at a very low level.
有几种基于聚合酶链反应（PCR）的特定方法可用于检测麻风分枝杆菌DNA，但扩增产物相当大。例如，常用于诊断的靶向36 kDa抗原基因的引物会产生一个530 bp的产物。在检测DNA可能已受损和片段化的样本时，这可能是一个缺点。因此，基于已被证明对麻风分枝杆菌具有特异性的现有引物对，设计了两组麻风分枝杆菌特异性巢式引物。靶向18 kDa抗原基因的引物产生136 bp的外侧产物和110 bp的内侧产物。基于RLEP重复序列的引物产生129 bp的外侧产物和99 bp的巢式产物。以标准麻风分枝杆菌灭活全细胞制剂的稀释液作为DNA来源，在优化实验条件后进行单阶段PCR和巢式PCR。与36 kDa抗原基因引物相比，18 kDa抗原基因外侧引物的灵敏度高100倍，RLEP外侧引物的灵敏度高1000倍。作为这些新引物两种可能应用的示例，从3例经治疗的瘤型麻风患者的皮肤切口样本和3例显示典型麻风古病理学的人类遗骸考古样本中获得了阳性结果。得出的结论是，这些新引物是检测受损或含量极低的麻风分枝杆菌DNA的有用手段。
