| Toxic mushroom has become one of the urgent food safety problems in China.In the research for poisonous mushrooms,more domestic and foreign research scholars focus on basidiomycetes and less on ascomycetes.Therefore,in this study,we conducted the identification technique research for ascomycete toxic mushrooms and their genera based on DNA bacording,DNA Mini-barcode,nucleotide signature and ring-mediated isothermal amplification techniques.In the aspect of DNA barcoding,we conducted DNA barcoding screening for Helvella,Peziza and Gyromitra based on evaluation of the success rate of intra-genus PCR amplification and sequencing,intra-and inter-species genetic variation,and species discrimination ability.Finally,ITS+LSU as core barcodes and ITS and LSU as auxiliary barcodes for Helvella were recommended;rpb2 as core barcodes and ITS and SSU as auxiliary barcodes for Peziza were recommended;ITS+SSU as core barcodes and ITS and SSU as auxiliary barcodes for Gyromitra were recommended.In order to solve the problem that full-length standard barcodes cannot amplify due to DNA degradation,we developed a DNA Mini-barcode for Gyromitra and designed its specific primers based on ITS sequence fragments.In order to solve the problem that the full-length standard barcode could not be amplified due to DNA degradation,we designed DNA Mini-barcode primers for Gyromitra based on ITS sequence fragments.The verification and analysis of versatility and applicability of the primers and species distinguishing analysis were conducted.The results showed that versatility and applicability of the Gyromitra primers were strong,which can accurately identify the samples with degradation DNA and solve the problem that the samples after boiled or digested cannot be identified by traditional morphology or standard DNA barcodes.Furthermore,the nucleotide signatures of Gyromitra Infula and Peziza vesicalosa with sequence lengths of 31 bp and 32 bp,respectively,were developed and rapid species detection techniques were explored based on the nucleotide signatures,specific primers were designed,and specification,applicability and sensitivity of the primers were verified and analyzed.The results showed that the nucleotide signatures of Gyromitra Infula and Peziza vesicalosa were species-unique,and the primers were highly specific and suitable for PCR amplification compared with the ITS universal primers,especially for the samples with severe DNA degradation.Gyromitra Infula and Peziza vesicalosa samples can be effectively detected with a sensitivity as low as 1 fg/μl,based on which the detection kits were developed in order to facilitate rapid and onsite detection.In addition,a rapid identification study of Gyromitra Infula and Helvella elastica was carried out based on ring-mediated isothermal amplification(LAMP).We designed LAMP primer sets for Gyromitra Infula and Helvella elastica based on interspecific differentiation and intraspecific conserved intervals,and performed primer specificity validation,sensitivity and applicability analyses.The results showed that Gyromitra Infula LAMP primer set could complete specific identification within 90 min and had high sensitivity with detection limit of 1ng;Helvella elastica LAMP primer set could complet specific identification within 60 min and had high sensitivity with detection limit of 1pg.The reaction results could be observed directly with naked eyes by colorimetric method,which provides an accurate,rapid and low-cost detection method for the identification of toxic mushroom species,and is suitable for onsite detection. |
