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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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Related in: MedlinePlus

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.
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pntd-0000328-g002: 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.

Mentions: After enumeration by direct microscopic counting, we extracted DNA for enumeration by RLEP TaqMan PCR using the highest enumerated sample of each tissue type to establish a standard curve for those tissues. As shown in Figure 2, direct microscopic counts ranged between 4.8×103 and 2.3×1010 bacilli. Estimates based on RLEP TaqMan PCR ranged from 623 organisms in conventional mouse foot pad tissues, to 5.8×1010 bacilli in each gram of armadillo tissue. For Molecular Enumeration, Coefficient of Variation (CV) between individual replicates averaged 14.03% (Mode 0.53%, Median 5.58%). Similar CV data was not available for the direct microscopic counts and no values were excluded based on CV. Enumeration estimates based on RLEP showed good correlation with direct microscopic counting with coefficients (Pearson's r) ranging from 0.78 to 0.89 for individual tissue types examined. Best results were seen with tissue sets that had a broad range of estimated bacillary counts. No significant difference in counting efficiency was seen between the various types (liver, spleen or lymph node) of armadillo tissues examined (data not shown). In combination across all tissues examined, RLEP showed a correlation of 0.98 (Pearson's) with direct microscopic counting.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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

pntd-0000328-g002: 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.
Mentions: After enumeration by direct microscopic counting, we extracted DNA for enumeration by RLEP TaqMan PCR using the highest enumerated sample of each tissue type to establish a standard curve for those tissues. As shown in Figure 2, direct microscopic counts ranged between 4.8×103 and 2.3×1010 bacilli. Estimates based on RLEP TaqMan PCR ranged from 623 organisms in conventional mouse foot pad tissues, to 5.8×1010 bacilli in each gram of armadillo tissue. For Molecular Enumeration, Coefficient of Variation (CV) between individual replicates averaged 14.03% (Mode 0.53%, Median 5.58%). Similar CV data was not available for the direct microscopic counts and no values were excluded based on CV. Enumeration estimates based on RLEP showed good correlation with direct microscopic counting with coefficients (Pearson's r) ranging from 0.78 to 0.89 for individual tissue types examined. Best results were seen with tissue sets that had a broad range of estimated bacillary counts. No significant difference in counting efficiency was seen between the various types (liver, spleen or lymph node) of armadillo tissues examined (data not shown). In combination across all tissues examined, RLEP showed a correlation of 0.98 (Pearson's) with direct microscopic counting.

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