Development and application of a new efficient and sensitive multiplex polymerase chain reaction (PCR) in diagnosis of leprosy.

ABSTRACT

India contributes about 80% of the global leprosy case load including case of fresh infection and reinfection. Due to lack of gold standard, diagnosis is done mainly based on routine clinical signs and symptoms, smear and histopathological evidences. There is a lot of lacunae in early confirmatory diagnosis in terms of sensitivity and specificity, especially in paucibacillary tuberculoid type. Moreover, the classification of different classes of leprosy is very important for selection of proper therapeutic schedule. Hence this study was undertaken to develop a multiplex polymerase chain reaction for the diagnosis and strain differentiation of M leprae. A multiplex polymerase chain reaction was developed using the primers R1 and R2 (a) amplifying 372bp DNA target from a repetitive sequence of M leprae and this repetitive sequence (372bp) that was used as a target DNA for amplification was reported to be specific for M leprae was not present in 20 mycobacterium species other than M leprae and primers TTCA and TTCB (b) amplifying (201bp) DNA target of variable sizes from the regions flanking TTC repeats of M leprae genome. This multiplex polymerase chain reacton developed in our laboratory revealed that the number of repeats at each locus might be variable among M leprae but they are found mostly in multibacillary (as the bacterial load is higher in multibacillary) type.