Development of a Loop-mediated isothermal amplification (LAMP) technique for specific and early detection of Mycobacterium leprae in clinical samples

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.

RLEP LAMP for the laboratory confirmation of leprosy: towards a point-of-care test

“…Four LAMPs for detecting M. leprae were published up to now (Garg et al, Jiang et al, Joshi et al and Joshi et al m-LAMP), all 100% specific, with a sensitivity ranging from 83 to 100% (being more sensitive for MB patients and tissue samples and less sensitive for PB patients and slit skin smears) and an analytical sensitivity of 30 bacilli each for both LAMPs from Joshi et al [ 51 – 54 ]. With a specificity of 100%, a sensitivity of 92% for minimal invasive samples (nasal swab samples and slit skin smears) and an analytical sensitivity of 27–43 bacilli (depending on the number of RLEP copies per strain) our results are well comparable with the other recently published assays.…”

“…With a specificity of 100%, a sensitivity of 92% for minimal invasive samples (nasal swab samples and slit skin smears) and an analytical sensitivity of 27–43 bacilli (depending on the number of RLEP copies per strain) our results are well comparable with the other recently published assays. The three published LAMPs applying standard consumables and easy-to-use nontechnical equipment have two limiting factors: non-specific amplification with different primers can generate false-positive samples, which is a common drawback of LAMP and—as described by Jiang et al—the fluorescence-based visual detection of results can be ambiguous, leading to the need of confirmation by gel electrophoresis, which would outweigh the benefits of the minimalist technique and can also generate false-positive samples due to contamination [ 51 – 53 ]. Joshi et al solved these problems with their m-LAMP in the same way as we did, i.e.…”

“…Four LAMP assays were recently designed for detecting M. leprae : Garg et al, Jiang et al and Joshi et al each designed RLEP or 16S LAMP assays, respectively, that required only standard consumables and a constant temperature water bath or heating block. Naked-eye detection was achieved via DNA intercalating dyes (colorimetric detection)—in case of Joshi et al with a closed tube technology to minimize the risk of cross-contamination—and additional visual detection of turbidity and bridge flocculation assay as used by Garg et al For further confirmation also agarose gel electrophoresis was used by Garg et al and Jiang et al [ 51 – 53 ]. Joshi et al additionally designed a RLEP multiplex LAMP (m-LAMP) for differential detection of M. leprae and Leishmania donovani using the portable real-time Genie ® II fluorometer from OptiGene (Horsham, United Kingdom) for amplification and detection of the LAMP products [ 54 ].…”

A low-cost, portable, and practical LAMP device for point-of-diagnosis in the field

“…LAMP technique has overcome majority of these limitations [2]. Improved and more sample-specific LAMP protocols have also been developed and published in recent decade [9][10][11][12][13][14][15][16][17][18][19]. In contrast to LAMP methods, technology development for LAMP-based detection has not been completely accelerated yet.…”

“…Hence, LAMP methods decrease cost, time and the need of expensive instruments or trained personnel. It has a great potential for breaking through the walls of laboratories to fields [9,10,11], clinics [12,13,14], farms [15,16,17], crime scenes [18,19] or even excavation sites.…”