CRISPR-Cas12a-assisted RT-RPA-LFA for rapid cluster IV-specific detection of cardamom mosaic virus in crude plant extracts.

Cardamom mosaic virus (CdMV), associated with mosaic/katte disease in cardamom, is a highly destructive virus that leads to severe reductions in plant growth and yield. High levels of genetic variability approaching species-level divergence exist among CdMV isolates from different geographical regions. Development of a cluster-specific diagnostic assay can enable reliable detection of locally prevalent virus populations and differentiate them from divergent ones. Therefore, in the present study, a CRISPR-Cas12a-coupled reverse transcription-recombinase polymerase amplification-lateral flow assay (RT-RPA-CRISPR-LFA) was developed for cluster IV-specific detection of Idukki isolates of CdMV. The viral target sequence (coat protein) was first amplified through RT-RPA using virus-specific primers designed from conserved region present in all strains of CdMV and crude plant extract template. The amplified product was subsequently recognized by CRISPR-Cas12a complex guided by a crRNA designed to specifically detect CdMV isolates associated with Idukki region of Kerala, triggering trans-cleavage activity of the complex and subsequent cleavage of the lateral flow reporter (LF reporter). This generated a visually detectable signal by LFA. The optimum assay performance was achieved with an RT-RPA incubation time of 10 min, LF reporter, Cas12a, and crRNA concentrations of 500, 75, and 125nM, respectively, and a CRISPR-Cas12a reaction incubation time of 1h. The assay was specific for CdMV strains representing Idukki region of Kerala and exhibited a detection limit of 1 fg (2.4 × 102 copies), which was 10 times less sensitive than qPCR. Practical feasibility and consistency of the assay were verified through validation using different field samples of cardamom.