Molecular Biology: Open Access

Validation of a Real-Time PCR (qPCR) Technique for Detection of Mycobacterium ulcerans in Clinical and Environmental Samples

Abstract

Tchalare Kondi Makagni, Issaka Maman, Eninam Kouma, Ebekalisai Piten, Yao Hoekou and Tchadjobo Tchacondo

Introduction: Buruli ulcer (BU) is a serious skin disease caused by Mycobacterium ulcerans. According to WHO, 70% of BU cases should be confirmed by the PCR-IS2404 gene amplification. The objective of this study is to validate a real-time PCR for the detection of M. ulcerans in clinical and environmental samples.
Methodology: A total of 70 clinical samples, 10 M. ulcerans strains and 15 environmental samples were tested by Ziehl-Neelsen staining technique, conventional PCR, real time qPCR and culture. The proportion of positive cases of M. ulcerans detection between the different tests was compared by the Chi-square test. The difference was
statistically significant for a P-value ≤ 0.05.
Results: Out of 33/80 samples were cultured positive (41.25%) to M. ulcerans, 41/80 ZN staining were positive (51.25%) to AFB at the microscopy, 55/80 (68.75%) and 64/80 samples (80%) were positive to the IS2404 insertion sequence from conventional PCR and qPCR respectively. Both PCR techniques showed positivity rates significantly
higher than microscopy and culture (P=0.002). However, no significant difference of positive rate was observed between the two types of PCR (P=0.38). In contrast, the detection limit for the real time qPCR was lower (0.01copies/ μL) compared to conventional PCR. Only qPCR was able to detect IS2404 in 2/15 environmental samples (13.3%).
Conclusion: This study showed the high sensitivity of the real time PCR for the detection of Mycobacterium ulcerans in clinical and environmental samples.