Enumeration of Mycobacterium leprae using real-time PCR

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.

Figure 1. RLEP TaqMan PCR results from titration of nude mouse-derived M. leprae.

Serial 2-fold dilutions of M. leprae were made from 2×10⁶ to 1.56×10⁴/ml. Ten microliters of each dilution were tested in triplicate representing 2×10⁴ to 156 M. leprae in the test sample, respectively. The ordinate is PCR cycle number at threshold and the abscissa is number of M. leprae (log₁₀). Standard deviations did not exceed 0.5% of mean at any dilution.

Figure 2. Comparison of direct microscopic counting of AFB per standard volume with enumeration of M. leprae by RLEP TaqMan PCR from tissues originating from a variety of host animals.

Symbols identify individual samples from sets of conventional, TNFR1 knock-out (KO), and congentially athymic nude mice, as well as from nine-banded armadillos. Pearson's coefficient (r²) is calculated for each tissue set. Enumeration estimates for all tissues combined showed high correlation (r² = 0.96) between the “gold standard' direct microscopic counting and estimates based on RLEP TaqMan PCR.

Figure 3. A comparison of RLEP TaqMan PCR and M. leprae counting results from a vaccine trial using conventional C57/B mice.

Bars represent mean plus the standard deviation for each group. ** = probability of statistical significance (p)<0.01, and *** = probability of statistical significance (p)<0.001.