Establishment of RPA-CRISPR/Cas12a-based visual detection of tomato mosaic virus
Tomato mosaic virus(ToMV)is a member of the genus Tobamovirus in the family Virgaviridae.ToMV mainly infects tomato,pepper,and most Solanaceae plants,resulting in severe losses in fruit quality and yield.In this study,the specific primers of recombinase polymerase amplification(RPA)and the crRNA of clustered regularly interspaced short palindromic repeats/CRISPR-associated 12a(CR1SPR/Cas12a)were designed based on the conserved sequence of the coat protein sequence of ToMV,and the reporter gene was selected.By optimizing the reaction system,a rapid visual detection method for ToMV detection was established,and the detection signal was strongest when the final concentration of fluorescent reporter FQ was 400 nmol/L,the Cas12a/crRNA ratio was 1∶5,and the final concentration was 200 nmol·L-1/1 000 nmol·L-1.With only 15 min of RPA and CR1SPR/Cas12a reaction,respectively,positive signals can be directly observed under a portable blue light irradiation equipment.This method can be used for specific detection of ToMV,and the minimal detection limit in detecting the total RNA of ToMV containing samples is 172 ag/μL,10 000 times that of RT-PCR-based ToMV detection.Hence,the established RPA-CRISPR/Cas12a-based detection is a rapid,sensitive and visual method for ToMV detection.
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