| Aflatoxins are carcinogenic and/or pathogenic toxins produced by aflatoxigenic fungi that often contaminate small grains, peanuts and feeds, threatening to human and animal health. Food or feed safety prohibits entry of contaminated products into market. However, current inspection methods, such as thin-layer chromatography, liquid chromatography, enzyme-linked immunosorbent assay and immunoaffinity column assay, are based on physical and chemical properties of the toxins and cannot be applied to examining risky products that may carry aflatoxigenic fungi but have not yet contained detectable toxins.Multiplex PCR reaction is a DNA-based detection method based on a regular PCR technique and can be used to identify simultaneously more DNA templates or different parts of the same DNA template in a single-lane reaction system. This method has been widely used in diagnosis of hazardous pathogens or microbes. In this study, several key enzyme genes involved in biological synthesis of the aflatoxins were used to design paired primers for developing muitiplex PCR detection system. The strain Aspergillus flavus AS 3.4408 was chosen as a positive aflatoxigenic fungus to testify sensitivity, reliability and efficiency of the developed system.Construction of multiplex PCR detection system. In an attempt to develop a multiplex PCR system for rapidly detecting aflatoxigenic fungi that often contaminate food and feed, four pairs of primers (i.e., ApaF/ApaR, OmtF/OmtR, VerF/VerR and ITS1/ITS4) were designed based on the sequences of three key genes (aflR, ver-1 and omt-1) involved in aflatoxin biosynthesis and of internal transcribed spacer (ITS) of fungal 5.8s rDNA. Amplified PCR products of the four target sequences fell in the sizes of 1032, 452, 797 and 600 bp, respectively, and were well in accordance with their DNA sequences documented in GenBank. The detection system was optimized for best reaction and successfully applied to detecting genomic DNA samples of six Aspergillus species and one Penicillium species. As a result, the four target DNA sequences were readily detected in three strains of Aspergillus flavus and A. parasiticus, which are known as aflatoxigenic fungi, whereas only ITS was found in the DNA samples of the rest unaflatoxigenic fungi. This shows that the developed multiplex PCR system had excellent specificity and sensitivity towards aflatoxigenic fungi. Further sensitivity analysis indicates that the developed system was featured with a conserved sensitivity of 1 ngμL-1 DNA sample, at which all the bands for the target DNA sequences were very clear. All the target sequences except aflR were also distinguishable even at the concentration of 0.1 ngμL-1 DNA sample.Improved extraction method of fungal DNA samples. Conventional methods of fungal DNA extraction, such as liquid nitrogen grinding, enzymolysis and TIANGEN? )extraction kit, were compared for their extraction efficiency. Based on the principles of the methods for breaking fungal cell walls, a novel rapid method for extracting genomic DNA from aflatoxigenic Aspergillus species was developed by making use of co-action of ultrasonic treatment and Tris-phenol-based lysis, followed by boiling-icing treatment and chloroform separation of the supernatant from residues. The resultant DNA samples suited well to the multiplex PCR detection. When this improved method was applied to extracting fungal DNAs, the PCR detection of samples potentially contaminated with aflatoxigenic fungi was completed with 5 h, meeting a rapid need for processing large samples.
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