Simultaneous identification of three clinically relevant peruvian pit vipers by multiplex loop-mediated isothermal...

Area of Science:

• Molecular Biology
• Herpetology
• Biotechnology

Background:

• Accurate identification of venomous snakes is crucial for managing ophidism (snakebite envenoming).
• Existing methods may not be rapid or field-deployable for distinguishing medically significant Peruvian species.

Purpose of the Study:

• To develop and validate a novel multiplex loop-mediated isothermal amplification (mLAMP) assay.
• To enable differential identification of three clinically relevant Peruvian venomous snakes: Bothrops atrox, Lachesis muta, and Crotalus durissus.

Main Methods:

• DNA extraction from shed snake skins.
• Amplification and sequencing of mitochondrial genes (Cytb, COI, 12S rRNA) for primer design.
• Development of species-specific primers and loop primers with fluorophores for mLAMP.
• Standardization of the mLAMP reaction with polyethyleneimine and detection via UV transillumination.

Main Results:

• Successful design and implementation of a multiplex assay.
• Differential fluorescence observed based on the snake DNA template.
• Assay demonstrated a detection limit of 0.2-25 ng DNA within 30 minutes.
• First report of mLAMP technology for venomous snake identification.

Conclusions:

• The developed mLAMP assay offers a rapid, sensitive, and specific method for identifying medically important Peruvian venomous snakes.
• This technology has potential applications in field diagnostics and epidemiological studies of ophidism.