Evaluation of Propidium Monoazide Real-Time PCR for Viablity of Mycobacterium leprae.
- Author:
Jong Pill KIM
1
;
Yeon Sil KIM
;
Chan Woo KIM
Author Information
1. Institute for Leprosy Research, Korean Hansen Welfare Association, Korea. dr_jpkim@hotmail.com
- Publication Type:Original Article
- Keywords:
Mycobacterium leprae;
Propidium monoazide;
Real-time PCR
- MeSH:
Animals;
DNA;
Foot;
Hot Temperature;
Mice;
Mycobacterium leprae*;
Mycobacterium*;
Polymerase Chain Reaction;
Propidium*;
Real-Time Polymerase Chain Reaction*;
ROC Curve;
Sensitivity and Specificity;
Suspensions
- From:Korean Leprosy Bulletin
2016;49(1):13-22
- CountryRepublic 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.
