Molecular Characterization of Mycobiota and Aspergillus Species from Eupolyphaga sinensis Walker Based on High-Throughput Sequencing of ITS1 and CaM

Abstract

Eupolyphaga sinensis Walker is a valuable traditional Chinese animal medicine first recorded in Shennong Bencao. Previous research has shown that E. sinensis is easily contaminated by aflatoxins (AFs), which are highly toxic mycotoxins, during harvest, storage, and transport, thereby posing a considerable threat to consumer health. Most often, these AFs are produced by Aspergillus species. In this study, we contrast the traditional culture-based dilution plating method to the high-throughput sequencing (HTS) technology for fungal identification in TCM E. sinensis. Both of the methods used internal transcribed spacer 1 (ITS1) and calmodulin (CaM) sequencing for fungal molecular identification. The new CaM primer we designed in the study is suitable for MiSeq PE300 sequencing used for identification of Aspergillus species in community DNA samples. More fungal species were found in the E. sinensis samples based on HTS than those found using the culture-based dilution plating method. Overall, combining the sequencing power of ITS1 and CaM is an effective method for the detection and monitoring of potential toxigenic Aspergillus species in E. sinensis. In conclusion, HTS can be used to obtain a large amount of sequencing data about fungi contaminating animal medicine, allowing earlier detection of potential toxigenic fungi and ensuring the efficient production and safety of E. sinensis.