Development of chikungunya virus detection by CRISPR-Cas12a-based detection

Abstract

Background: The chikungunya virus is a mosquito-borne virus that causes acute fever with potentially prolonged joint pain. Aedes aegypti and Aedes albopictus are primary vectors for chikungunya in Asia, as well as dengue and Zika viruses. The viruses co-circulate in the same habitat and cause a similar acute fever in early onset, posing a misdiagnosis challenge. The reverse transcription-polymerase chain reaction is the only diagnostic option for detecting the chikungunya viral gene.

Objective: This study aimed to evaluate the designed primers and crRNAs for a molecular assay of chikungunya viral gene detection using RT-RPA amplification coupled with a CRISPR-Cas12a detection system.

Methods: Chikungunya virus complete genome sequences available from National center for biotechnology information (NCBI) were aligned using Jalview to identify potential crRNAs and primers in conserved regions. The designed recombinase polymerase amplification (RPA) primers and crRNAs were validated to determine the optimal primers and crRNAs.

Results: Following the alignment of Chikungunya viral genome sequences, primers and crRNAs were designed for the nsP2 gene, which is a conserved region suitable for detection. The designed primers and crRNAs were successfully used in CHIK gene amplification and detection.

Conclusion: This study offers a molecular assay that could be a potential diagnostic test for chikungunya viral detection. Further investigations include evaluation for the limit of detection, cross-reactivity, and clinical sensitivity and specificity tests.