Korean Leprosy Bulletin  2016;49(1):13-22

Evaluation of Propidium Monoazide Real-Time PCR for Viablity of Mycobacterium leprae.

Jong Pill KIM 1 ; Yeon Sil KIM ; Chan Woo KIM

Affiliations

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Keywords

Mycobacterium leprae; Propidium monoazide; Real-time PCR

Country

Republic of Korea

Language

Korean

Abstract

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.