An Efficient Droplet Digital PCR Approach for Detection DNA at Low Concentrations of Toxigenic Fungi in Food Products

As common toxigenic fungi genera, Aspergillus , Penicillium , and Fusarium , have attracted the attention of governments and people all over the world due to their human carcinogenicity, teratogenicity, and hepatotoxicity. Accordingly, effective and quantitative detection method for toxigenic fungi before mycotoxins produced should be established to ensure food safety. In this paper, two duplex-droplet digital PCR (ddPCR) were developed and optimized for toxigenic fungi. The detection limits of the target genes, including the AflR , Och , Pen , and Fus of toxigenic fungi, were 26 copies/reaction, 15 copies/reaction, 161 copies/reaction, and 29 copies/reaction, respectively. Notably, the detection limit of duplex ddPCR was three orders of magnitude higher than that of tradition real-time fluorescence PCR reaction. Moreover the efficiency and sensitivity of the established method were also higher than those of real-time fluorescence PCR. The linear quantitative range of copy number of AflR and Och genes in AflR/Och duplex system and Pen and Fus genes in Pen / Fus duplex system were both 2 × 10 −7 –2 × 10 −3 ng/μL. From DNA extraction to target gene detection, time consume of 96 samples was within 6 h, achieving the purpose of high-throughput and rapid detection.