สถาบันวิจัยวิทยาศาสตร์สาธารณสุข

NATIONAL INSTITUTE OF HEALTH OF THAILAND

LightCycler real-time PCR for quantitative detection of Mycobacterium leprae

Authors : Janisara Rudeeanaksin*, Sopa Srisungngam*, Taweerit Sithivekin**, Krisada Mahotan**, Patrick J. Brennan***, Prasit Palittapongarnpim****, and Benjawan Phetsuksiri*

 

Affiliations:    * Thai National Institute of Health, Department of Medical Sciences, Ministry of Public Health, Nonthaburi, Thailand 
** Raj-Prachasamasai Institute, Department of Disease Control, Ministry of Public Health, Nonthaburi, Thailand
*** Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, Colorado, USA
                                **** Department of Microbiology, Mahidol University, Bangkok Thailand
 
Source:         Proceeding: 10th Asean Conference in Medical Laboratory Technology 2004; 264
 
Language:      English
 
Abstract:
 
Enumeration of Mycobacterium leprae for determination of bacterial index is required for leprosy classification, monitoring of leprosy chemotherapy and diagnosis of relapse. In clinical application, quantification of M. leprae relies on microscopic examination and counting of bacilli in stained specimens. The counting method yielding results with limited specificity and sensitivity. This study is aimed to develop and evaluate the application of a fluorescence real-time PCR assay for quantifying M. leprae in skin specimens. The real-time PCR is based on a capillary format of the LightCycler using SYBR Green I fluorescent dye as a detection signal. Primers were applied to amplify a portion of 171 bp fragment of M. leprae 16S rRNA gene. Using commercial Flexigene, with modifications, resulted in high yields of isolated DNA. The PCR assay was specific for M. leprae and able to detect as low as 20 fg of M. leprae DNA. The analytical sensitivity was as low as one cell of bacilli. The melting temperature of this PCR product was 86°C. By the use of normalized quantitative real-time PCR, M. leprae was detected in 40 multibacillary (MB) patients with bacilli number in the range of 9.6 x 102 - 8.5 x 107 bacilli in 6x6 mm skin biopsy specimen. The detectable number of bacilli in skin biopsies from 10 paucibacillary (PB) patients is of the order of 8.9 x 102 - 2.5 x 103 bacilli. The preliminary results demonstrated thatLightCycler real-time PCR appeared to be a robust tool for quantitative detection of leprosy bacilli in clinical specimens and could be adopted as a molecular tool for quantification of M. leprae in other experimental settings.