Multiplex PCR Identification of Aspergillus cristatus and Aspergillus chevalieri in Liupao Tea Based on Orphan Genes

“Golden flower” fungi in dark tea are beneficial to human health. The rapid identification method of “golden flower” fungi can verify the quality of dark tea products and ensure food safety. In this study, 6 strains were isolated from Liupao tea. They were respectively identified as A. cristatus, A. chevalieri, and A. pseudoglaucus. A. pseudoglaucus was reported as Liupao tea “golden flower” fungus for the first time. It was found that the ITS and BenA sequences of A. cristatus and A. chevalieri were highly conserved. It is difficult to clearly distinguish these closely related species by ITS sequencing. To rapidly identify species, multiplex PCR species-specific primers were designed based on orphan genes screened by comparative genomics analysis. Multiplex PCR results showed that orphan genes were specific and effective for the identification of A. cristatus and A. chevalieri isolated from Liupao tea and Fu brick tea. We confirmed that orphan genes can be used for identification of closely related Aspergillus species. Validation showed that the method is convenient, rapid, robust, sequencing-free, and economical. This promising method will be greatly beneficial to the dark tea processing industry and consumers.