PCR primers that can detect low levels of Mycobacterium leprae DNA

Abstract

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.