A CRISPR-Cas12a-Based Diagnostic Method for Japanese Encephalitis Virus Genotypes I, III, and V

Area of Science:

• Molecular biology
• Virology
• Infectious diseases

Background:

• Japanese encephalitis virus (JEV) is a significant public health concern in Asia, transmitted by Culex mosquitoes.
• Current prevention relies on vaccination, with no approved antiviral treatments available for JEV infection.
• CRISPR-Cas12a-based diagnostics, like DETECTR, offer novel molecular detection capabilities.

Purpose of the Study:

• To evaluate the efficacy of DETECTR combined with reverse transcription-recombinase polymerase amplification (RT-RPA) for JEV diagnosis.
• To determine the sensitivity and specificity of this novel diagnostic approach for JEV genotypes.
• To develop and assess a simplified one-tube, one-temperature format of DETECTR for JEV detection.

Main Methods:

• Utilized DETECTR coupled with RT-RPA for the detection of JEV RNA.
• Assessed the limit of detection for different JEV genotypes (GI, GIII, GV).
• Compared the performance of a one-tube, one-temperature DETECTR format against conventional DETECTR.

Main Results:

• DETECTR with RT-RPA detected JEV down to 10 RNA copies for GI and 1 x 10^2 copies for GIII and GV.
• The method demonstrated comparable sensitivity to RT-PCR and showed no cross-reactivity with other viruses.
• The one-tube, one-temperature format maintained diagnostic efficiency.

Conclusions:

• DETECTR combined with RT-RPA is a highly sensitive and specific molecular diagnostic tool for JEV.
• This method offers a viable alternative for JEV detection, particularly in resource-limited settings.
• Further development of simplified formats like the one-tube system enhances its practical application.