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KMID : 0386320160490010013
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Korean Leprosy Bulletin
2016 Volume.49 No. 1 p.13 ~ p.22
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Evaluation of Propidium Monoazide Real-Time PCR for Viablity of Mycobacterium leprae
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Kim Jong-Pill
Kim Yeon-Sil
Kim Chan-Woo
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Abstract
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Background : Conventional acid-fast bacilli (AFB) staining cannot differentiate viable from dead cells. Propidium monoazide (PMA) is a photoreactive DNA-binding dye that inhibits PCR amplification by DNA modification.
Object : The author evaluated whether PMA real-time PCR is suitable for the viablity of Mycobacterium leprae ( M. leprae) in specimens of cultivation in mouse foot pads.
Methods : A total of 55 diluted suspensions from mouse foot pads were quadruplicated and subjected to PMA treatment and/or heat inactivation, and were also tested to compare the ΔCT values (CT value in PMA-treated samples-CT value in non- PMA-treated samples). Real-time PCR was performed using QuantiTect SYBR® Green PCR Kits(Qiagen, USA), and the CT value changes after PMA treatment were compared between PMA treatment and/or heat inactivation groups.
Results : The increase in the CT value after PMA treatment was significant in heat inactivated group(4.26) and non-heat inactivated group(1.12)(both P = 0.000). In the ROC curve analysis, the cutoff ΔCT value for maximum sensitivity (100%) and specificity (97.1%) for differentiating dead from live cells was 2.41.
Conclusions : PMA real-time PCR is a useful approach for evaluating viablity of M. leprae.
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KEYWORD
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Mycobacterium leprae, Propidium monoazide, Real-time PCR
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