The invention belongs to the field of
agriculture and
plant quarantine, and particularly relates to a method for detecting
phomopsis helianthi based on RPA /
CRISPR-Cas12a, which comprises the following steps: S1, designing a detection composition: designing a composition for detecting
phomopsis helianthi, the composition comprising a specific primer RPA-F / R
nucleic acid sequence, a specific crRNA
nucleic acid sequence and probes FL-ssDNA and LF-ssDNA; s2, extracting
DNA: extracting the
genome DNA of the diseased tissue of the
sunflower to be detected; s3,
DNA isothermal amplification: taking the
genome DNA obtained in the step S1 as a template, adding a specific primer RPA-F1 / R1 into the template, and carrying out RPA amplification to obtain an amplification product; s4,
CRISPR / Cas12a reaction: the amplification product obtained in the S2 and the crRNA are respectively mixed with FL-ssDNA and LF-ssDNA probes for a cleavage reaction, and a
reaction product FL and a
reaction product LF are obtained; and S5,
visual detection: respectively placing the
reaction product FL and the reaction product LF in a
fluorescent PCR instrument and on a Cas12 special detection test strip for detection. The method is based on an RPA /
CRISPR-Cas12a technology, by designing a specific primer and a crRNA
nucleic acid sequence, the detection duration of the
phomopsis helianthi is greatly shortened, the detection sensitivity is improved, and the method has very high use value in detection scenes such as customs and the like.