Korean Lepr Bull.  2016 Dec;49(1):13-22. 10.0000/klb.2016.49.1.13.

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

• Kim JP
• Kim YS
• Kim CW

• KMID: 2369142
• DOI: http://doi.org/10.0000/klb.2016.49.1.13

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.

Keyword

Mycobacterium leprae; Propidium monoazide; Real-time PCR

MeSH Terms

Animals
DNA
Foot
Hot Temperature
Mice
Mycobacterium leprae*
Mycobacterium*
Polymerase Chain Reaction
Propidium*
Real-Time Polymerase Chain Reaction*
ROC Curve
Sensitivity and Specificity
Suspensions
DNA
Propidium
Suspensions