Rift Valley fever virus M and L genome segment detection: A comparison of field deployable reverse transcription insulated isothermal PCR (RT-iiPCR) and laboratory based multiplex reverse transcriptase real-time PCR

Authors: TRUJILLO, JESSIE - Kansas State University; Wilson, William - Bill; CRAIG, ANTHONY - University Of Pretoria; VAN DEN BERGH, CARIEN - Kansas State University; WANG, THOMAS - Genereach Usa; THOMPSON, PETER - University Of Pretoria; SWANEPOEL, ROBERT - University Of Pretoria; MOROZOZ, IGOR - Kansas State University; RICHT, JUERGEN - Kansas State University

Submitted to: Journal of Clinical Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/21/2023
Publication Date: 2/2/2024
Citation: Trujillo, J.D., Wilson, W.C., Craig, A., Van Den Bergh, C., Wang, T., Thompson, P.N., Swanepoel, R., Morozoz, I., Richt, J. 2024. Rift Valley fever virus M and L genome segment detection: A comparison of field deployable reverse transcription insulated isothermal PCR (RT-iiPCR) and laboratory based multiplex reverse transcriptase real-time PCR. Journal of Clinical Microbiology. 63(3). https://doi.org/10.1128/jcm.00430-23.
DOI: https://doi.org/10.1128/jcm.00430-23

Interpretive Summary: The mosquito-borne Rift Valley fever phlebovirus (RVFV) causes major agricultural and public health problems in Africa and is considered a potential agro-bioterrorism agent with limited countermeasures. This paper describes a rapid, and sensitive molecular method for point of care (POC) or point of need (PON) that can be deployed to field applications. The strategy involves detection of the RVFV genome on a portable, touch screen instrument. The system sensitivity and specificity were tested in the laboratory then evaluated in the two endemic African countries using ruminant sera. The system provides a tool that is reliable, sensitive and specific for point of need RVFV genomic detection that could be used in diagnostic, surveillance and epidemiological applications.

Technical Abstract: Rift Valley fever phlebovirus (RVFV) is a mosquito-borne pathogen that causes major agricultural and public health problems in Africa and the Arabian Peninsula. It is considered a potential agro-bioterrorism agent with limited countermeasures. To address diagnostic needs, we describe a novel, rapid, and sensitive molecular method capable of immediate deployment at sites of suspected outbreaks. The strategy involves concurrent detection of two of the three RVFV genome segments (L and M) using reverse transcriptase insulated isothermic PCR (RT-iiPCR) performed on a portable, touch screen nucleic acid analyzer, POCKITTM. The analytical sensitivity for both the RT-iiPCR and a laboratory-based L and M multiplex RT-qPCR assay was estimated at approximately 0.1-3 copies/reaction using synthetic RNA or viral RNA. The diagnostic sensitivity and specificity of detection of RVFV on POCKITTM, determined using sera from sheep and cattle (n=175) experimentally infected with two strains of RVFV (SA01 and Ken06) was 93.8% and 100% (kappa= 0.93), respectively. Testing of ruminant field sera (n=191) in two locations in Africa demonstrated 100% diagnostic sensitivity and specificity. We conclude that the POCKITTM dual gene RVFV detection strategy can provide reliable, sensitive and specific point of need viral RNA detection. Moreover, field detection of RVFV in vectors or susceptible animal species could aid in surveillance and epidemiological studies to better understand and control RVFV outbreaks.