Construction method and application of multiple PCR (Polymerase Chain Reaction) reaction system for detection and species identification of coyal spot pathogen

The invention discloses a construction method and application of a multiplex PCR (Polymerase Chain Reaction) reaction system for detection and species identification of colletotrichum aleosporioides. The method comprises the following steps: obtaining whole genome sequences of a plurality of species of colletotrichum aleosporioides, and carrying out multi-sequence comparison to obtain specific genes and specific gene clusters of the species; a plurality of upstream primers and downstream primers are designed according to a conventional primer design method by taking genes in specific gene clusters of various species as templates, primer pairs are generated, and unreasonable primer pairs with low sensitivity and specificity are excluded. According to the size of an amplification product, the primers are freely combined and matched to generate a multiple primer formed by mixing four pairs of primers, and multiple PCR reaction conditions are optimized to obtain a multiple PCR system capable of specifically detec