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Enumeration of Mycobacterium leprae using real-time PCR.

Truman RW, Andrews PK, Robbins NY, Adams LB, Krahenbuhl JL, Gillis TP - PLoS Negl Trop Dis (2008)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Health and Human Services, Health Resources Services Administration, Bureau of Primary Health Care, National Hansen's Disease Programs, Baton Rouge, Louisiana, USA.

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.

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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.
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pntd-0000328-g003: 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.

Mentions: To determine the effect of host resistance towards M. leprae on the efficiency of molecular enumeration, we compared counting results obtained with the two techniques in a standard mouse foot pad (MFP) vaccine study. The seminal work of Shepard, et al [24] and a large number of subsequent studies have demonstrated the suppressive effect of potent vaccines, such as BCG and heat-killed M. leprae, on the growth of M. leprae in the mouse foot pad model. Briefly, prior sensitization with heat-killed M. leprae will result in 1 to 2 logs growth suppression in the mouse foot pad. In our studies mice were vaccinated with heat-killed M. leprae (HKML) or saline, as a sham vaccine, and challenged with 5000 viable M. leprae in their foot pads thirty days following vaccination. M. leprae enumerations were performed 6 months after challenge by RLEP TaqMan PCR and direct microscopic counting. The results of the vaccine trial are shown in Figure 3.


Enumeration of Mycobacterium leprae using real-time PCR.

Truman RW, Andrews PK, Robbins NY, Adams LB, Krahenbuhl JL, Gillis TP - PLoS Negl Trop Dis (2008)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2570796&req=5

pntd-0000328-g003: 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.
Mentions: To determine the effect of host resistance towards M. leprae on the efficiency of molecular enumeration, we compared counting results obtained with the two techniques in a standard mouse foot pad (MFP) vaccine study. The seminal work of Shepard, et al [24] and a large number of subsequent studies have demonstrated the suppressive effect of potent vaccines, such as BCG and heat-killed M. leprae, on the growth of M. leprae in the mouse foot pad model. Briefly, prior sensitization with heat-killed M. leprae will result in 1 to 2 logs growth suppression in the mouse foot pad. In our studies mice were vaccinated with heat-killed M. leprae (HKML) or saline, as a sham vaccine, and challenged with 5000 viable M. leprae in their foot pads thirty days following vaccination. M. leprae enumerations were performed 6 months after challenge by RLEP TaqMan PCR and direct microscopic counting. The results of the vaccine trial are shown in Figure 3.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Health and Human Services, Health Resources Services Administration, Bureau of Primary Health Care, National Hansen's Disease Programs, Baton Rouge, Louisiana, USA.

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.

Show MeSH
Related in: MedlinePlus