Date Published: November 4, 2008
Publisher: Public Library of Science
Author(s): Richard W. Truman, P. Kyle Andrews, Naoko Y. Robbins, Linda B. Adams, James L. Krahenbuhl, Thomas P. Gillis, Pamela L. C. Small
Abstract: Mycobacterium leprae is not cultivable in axenic media, and direct microscopic enumeration of the bacilli is complex, labor intensive, and suffers from limited sensitivity and specificity. We have developed a real-time PCR assay for quantifying M. leprae DNA in biological samples. Primers were identified to amplify a shared region of the multicopy repeat sequence (RLEP) specific to M. leprae and tested for sensitivity and specificity in the TaqMan format. The assay was specific for M. leprae and able to detect 10 fg of purified M. leprae DNA, or approximately 300 bacteria in infected tissues. We used the RLEP TaqMan PCR to assess the short and long-term growth results of M. leprae in foot pad tissues obtained from conventional mice, a gene knock-out mouse strain, athymic nude mice, as well as from reticuloendothelial tissues of M. leprae–infected nine-banded armadillos. We found excellent correlative results between estimates from RLEP TaqMan PCR and direct microscopic counting (combined r = 0.98). The RLEP TaqMan PCR permitted rapid analysis of batch samples with high reproducibility and is especially valuable for detection of low numbers of bacilli. Molecular enumeration is a rapid, objective and highly reproducible means to estimate the numbers of M. leprae in tissues, and application of the technique can facilitate work with this agent in many laboratories.
Partial Text: Because M. leprae can not be grown on synthetic media, the bacilli must be enumerated by direct microscopic counting. Originally developed by Shepard  in the 1960’s, this technique has survived as the “gold standard” for enumerating M. leprae for almost 50 years. Unfortunately, it is a highly specialized procedure, cumbersome to perform and limited in terms of sensitivity and specificity. Only a few laboratories today have retained the ability to enumerate M. leprae using direct microscopy ,.
These results demonstrate that a simple, reproducible test based on genomic DNA can be used to quantify M. leprae in infected tissues. The real time PCR assay yields results similar to those obtained from conventional direct microscopic counting methods, is highly specific, sensitive, and is easily adapted to large scale batch processing of samples. Molecular quantification of M. leprae based on amplification of RLEP TaqMan PCR is a suitable replacement for direct microscopic counting of bacilli.