Indian J Lepr 2008, 80 : 315-321 http://www.ijl.org.in
Original Article

Development and Evaluation of Real-Time RT-PCR
Assay for Quantitative Estimation of
Viable Mycobacterium leprae in Clinical Samples


R Sharma1, M Lavania1, K Katoch2, DS Chauhan1,
AK Gupta1, UD Gupta3, VS Yadav4, VM Katoch5
 


    1R Sharma, PhD, Research Scholar, Department of Microbiology and Molecular Biology
    1M Lavania, MSc, Research Scholar, Department of Microbiology and Molecular Biology
    1DS Chauhan, PhD, Scientist C, Department of Microbiology and Molecular Biology
    1 AK Gupta, MSc, Research Scholar, Department of Microbiology and Molecular Biology
    2 K Katoch, MD, Scientist F and Head, Medical Unit-I; Model Rural Health Research Unit, Ghatampur, Kanpur, India
    3UD Gupta, PhD, Scientist E, Laboratory for Animal Experiments
    4VS Yadav, MA (Stat), Research Assistant, Department of Biostatistics
    National JALMA Institute for Leprosy and Other Mycobaceterial Diseases (ICMR), Dr M Miyazaki Marg, Tajganj, Agra- 28200 I, India.

    5VM Katoch, MD, Secretary, Department of Health Research, Ministry of Health and Family Welfare, Govt of India and Director-General, Indian Council of Medical Research, Ansari Nagar, New Delhi-110029, India.


    Correspondence to : Dr VM Katoch, Email : vishwamohan_katoch@yahoo.co.in




Abstract


Detection of live organisms by molecular methods has special significance in leprosy where causative organism can not be cultivated in vitro. Such techniques would be especially important for monitoring the progress of the disease. While real-time RT- PCR technology will be appropriate for this purpose, there is very little experience of use of such tools in leprosy. This study describes the development of a quantitative RT-PCR targeting 16S rRNA based on primers used in a semi quantitative RT-PCR and its application on clinical samples including slit scraping and biopsies. RNA was extracted from biopsies from 3 lepromatous leprosy (LL) cases and standard curve was generated by plotting crossing over point against the dilutions of input RNA quantity (number of bacilli used for RNA extraction). Real-time RT-PCR was performed for quantitative detection of live M.leprae in 28 slit (13/28 smear positive) scrappings and 32 biopsies (22/32 smear positive). Number of viable bacteria as estimated by solid stained bacilli and real-time PCR correlated (no difference p>0.05). The test achieved a theoretical analytical sensitivity limit of up to single live bacillus even considering 11.3% efficiency of RNA preparation which was calculated by spiking of known number of leprosy bacilli in non leprosy skin biopsies (PCR negative). All smear positive cases were positive by this assay. This assay appears to be a promising tool for detection and quantification of viable bacilli in selected clinical situations and should be of use even in smear negative cases also.

Key words : Quantitative estimation, Real-time RT-PCR, M. leprae, Clinical Sample