On-site and visual detection of sorghum mosaic virus and rice stripe mosaic virus based on reverse transcription-recombinase-aided amplification and CRISPR/Cas12a.

Abstract

Rapid, sensitive and visual detection of plant viruses is conducive to effective prevention and control of plant viral diseases. Therefore, combined with reverse transcription and recombinase-aided amplification, we developed a CRISPR/Cas12a-based visual nucleic acid detection system targeting sorghum mosaic virus and rice stripe mosaic virus, which cause harm to crop production in field. When the RT-RAA products were recognized by crRNA and formed a complex with LbCas12a, the ssDNA labeled with a quenched green fluorescent molecule will be cleaved by LbCas12a, and then a significant green fluorescence signal will appear. The entire detection process can be completed within 30 min without using any sophisticated equipment and instruments. The detection system could detect samples at a dilution of 10⁷, about 10⁴-fold improvement over RT-PCR, so the system was successfully to detect rice stripe mosaic virus in a single leafhopper, which is the transmission vector of the virus. Finally, the CRISPR/Cas12a-based detection system was utilized to on-site detect the two viruses in the field, and the results were fully consistent with that we obtained by RT-PCR in laboratory, demonstrating that it has the application prospect of detecting important crop viruses in the field.