Development and Validation of Rapid Colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification for Detection of Rift Valley Fever Virus

Abstract

Rift Valley fever virus (RVFV) is a high-priority zoonotic pathogen with the ability to cause massive loss during its outbreak within a very short period of time. Lack of a highly sensitive, instant reading diagnostic method for RVFV, which is more suitable for on-site testing, is a big gap that needs to be addressed. Te aim of this study was to develop a novel one-step reverse transcription loop-mediated isothermal amplifcation (RT-LAMP) method for the rapid detection of RVFV. To achieve this, the selected RVFV M segment nucleotide sequences were aligned using Multiple Sequence Comparison by Log-Expectation (MUSCLE) software in MEGA11 version 11.0.11 program to identify conserved regions. A 211 pb sequence was identifed and six diferent primers to amplify it were designed using NEB LAMP Primer design tool version 1.1.0.Te specifcity of the designed primers was tested using primer BLAST, and a primer set, specifc to RVFV and able to form a loop, was selected. In this study, we developed a single-tube test based on calorimetric RT-LAMP that enabled the visual detection of RVFV within 30 minutes at 65°C. Diagnostic sensitivity and specifcity of the newly developed kit were compared with RVFV qRT-PCR, using total RNA samples extracted from 118 blood samples. Te colorimetric RT-LAMP assay had a sensitivity of 98.36% and a specifcity of 96.49%.Te developed RT-LAMP was found to be tenfold more sensitive compared to the RVFV qRT-PCR assay commonly used in the confrmatory diagnosis of RVFV.