葡萄灰霉病菌LFD-RPA检测方法的建立与评价OA

Gray mold,caused by infection with Botrytis cinerea,is one of the major diseases in grape cultivation.This study developed a rapid and precise detection approach employing recombinase polymerase amplification in conjunction with a lateral flow dipstick(LFD-RPA),based on the Bc-hch gene sequence of B.cinerea(GenBank accession no.OQ411027.1)Specific primers(FRA-F/FRA-R)and a probe(LF-P)were designed,and reaction conditions were optimized.The method's sensitivity and accuracy were validated against quantitative real-time PCR(qRT-PCR)and conventional PCR.Results showed that FRA-F/FRA-R and LF-P exhibited high specificity.The LFD-RPA assay detected B.cinerea within 30 minutes at 37℃,producing visible bands on the dipstick.Its detection limit was 10-1ng·μL-1,similar to conventional PCR but slightly lower than qRT-PCR.Field sample testing showed 100％consistency between LFD-RPA,conventional PCR,and qRT-PCR,domastrating its accuracy.All three methods detected B.cinerea during the latent infection stage,as confirmed by tests at various post-inoculation time points.The LFD-RPA assay provides a reliable and rapid tool for monitoring gray mold in grapevines.