Standardization of the technique 'LOOP-MEDIATED ISOTHERMAL AMPLIFICATION' (LAMP) using the target 18S rDNA for cutaneous leishmaniasis v2

Leishmaniasis are infectious diseases, transmitted by vectors, caused by protozoa of thegenus Leishmania. Several protocols for molecular testing (conventional PCR -cPCR; and real-time PCR - qPCR) have been evaluated for the diagnosis ofcutaneous leishmaniasis (CL), but they are limited to reference centers. Abreakthrough in the diagnosis of various infectious diseases has been thedevelopment of the technique 'loop-mediated isothermal amplification' - LAMP.This method is capable of amplifying large amounts of DNA under isothermalconditions within 30-60 minutes. Our goal was toevaluate the performance of the LAMP technique targeting 18S rDNA using theWarmStart‱ Colorimetric LAMP 2x Master Mix (WS) kit and the Bst 2.0 DNAPolymerase enzyme with SYBR‱ Green I (BstSg). The analyticalsensitivity (detection limit) with the WS mix was up to 10 fg in agarose geland 100 fg visually, with consistent results in duplicates; and using BstSg,the sensitivity was up to 10 pg. In the analytical specificity using WS,there was reactivity for all Leishmania samples. However, the samples ofParacoccidioides brasiliensis (Pb) and Sporothrix globosa (Sg)showed bands in one of the duplicates in the gel, suggesting possiblenon-specific amplification. Using BstSg, visual reactivity was observed onlyfor L. braziliensis (Lb). However, in agarose gel, samples of L.infantum (Li), L. guyanensis (Lg), and one of the duplicates of L.amazonensis (La) showed bands suggestive of amplification. The LAMP is a promising alternative for the diagnosis of leishmaniasis.