Abstract
Leprosy, a progressive, mutilating and highly stigmatized disease caused by Mycobacterium leprae (ML), continues to prevail in the developing world. This is due to the absence of rapid, specific and sensitive diagnostic tools for its early detection since the disease gets notified only with the advent of physical scarring in patients. This study reports the development of a Loop-mediated isothermal amplification (LAMP) technique for fast, sensitive and specific amplification of 16S rRNA gene of ML DNA for early detection of leprosy in resource-limited areas. Various parameters were optimized to obtain robust and reliable amplification of ML DNA. Blind clinical validation studies were performed which showed that this technique had complete concurrence with conventional techniques. Total absence of amplification of negative control DNA confirmed the specificity of this test. Various visual detection methods viz. colorimetric, turbidity differentiation and bridge flocculation were standardized to establish easy-to-read and rapid diagnosis. This technique eliminates the lack of accuracy and sensitivity in skin smear tests in patients and the requirement for expensive lab equipments and trained technicians. The technique holds promise for further expansion and has the potential to cater to the unmet needs of society for a cheap, highly-sensitive and robust rapid diagnosis of ML.
Highlights
We report the development and standardization of a rapid, sensitive and economical method of detecting Mycobacterium leprae (ML) DNA using an optimized loop-mediated isothermal amplification technique (LAMP) technique
The challenges in identification and recruitment of patients in early stages of ML infection remain high, expanding the clinical sample size in future studies will help in eliminating any possible bias or inaccuracy in our proposed technique
The technique is suitable for detecting ML DNA even at picogram concentrations, making it highly sensitive to low bacterial load and can be applied for early diagnosis of the disease before any permanent physical mutilation
Summary
Isothermal amplification methods are favored over traditional PCR methods for developing cheap, effective and rapid diagnostics as the requirement of expensive laboratory equipments like thermocyclers is eliminated, without compromising efficiency or sensitivity. DNA amplification can be completed in a single step by incubating the mixture of primers with polymerase and template in a reaction buffer using a simple heat block or water bath[18] It can typically produce results with high yield and specificity in less than 30 min at a single temperature. For on-field, simple, rapid and user-friendly endpoint detection, various amplicon detection methods have been explored by researchers that can be combined with LAMP technique to produce immediate and easy to read results. Current study is the first report on presenting LAMP as an efficient diagnostic tool for ML
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