Simultaneous identification of three clinically relevant peruvian pit vipers by multiplex loop-mediated isothermal amplification (mLAMP)

Abstract

Previous knowledge about the taxonomic distribution of venomous snake species is very useful for epidemiological aspects of ophidism. Here, we sought to develop an assay for the differential identification of clinically relevant snakes in Peru: Bothrops atrox, Lachesis muta, and Crotalus durissus using a multiplex loop-mediated isothermal amplification (mLAMP) assay. For this, DNA was extracted from the shed snake skins and the mitochondrial genes Cytb, COI, and 12S rRNA were amplified and further sequenced, for the design of mLAMP reaction primers. For each snake species the forward and reverse primers, internal forward and reverse primers, and the loop primers were obtained, bearing the latter different fluorophores for product identification. Finally, the reaction was standardized in the presence of all primer sets, and an optimal amount of low molecular weight polyethyleneimine. The precipitated products were observed in a UV light transilluminator, finding a differential fluorescence according to the DNA used, with a detection limit to the naked eye in the range of 0.2-25 ng of DNA, within 30 min. This study is the first report on the use of mLAMP technology for the identification of venomous snakes.

Keywords: Bothrops atrox; Crotalus durissus; Lachesis muta; Mitochondrial DNA; Snake; mLAMP.