Screening Of Standard Genes In Wild Edible Fungus Matsutake And Research On Constant Temperature Detection Technology

At present,Tricholoma matsutake is a nutrient-rich wild edible fungus,however,the origins are is limited,which causes the increase of the adulteration of Tricholoma matsutake.The traditional detection methods depending on morphology have many limitations.With the widespread applications of modern molecular biology techniques in food science field,the molecular biology techniques in quick identifying the adulteration of wild Tricholoma matsutake and its deep processed products have a good developing prospect.In this study,the endogerous reference gene of Tricholoma matsutake was screen,and the specific primers were designed and verification.The Loop-mediated isothermal amplification(LAMP)reaction system,including seven correlation factors,were optimized,and then the qualitative and quantitative LAMP detection methods were established.The Gold immunochromatography assay(GICA)was combined with the LAMP,by optimizing the detection time and buffer solution,a fast and visual LAMP-colloidal gold nucleic acid dipstick assay was developed.The main research results were as follows:1.The genomic DNA of Yunnan Shangri-La Tricholoma matsutake was isolated by CTAB,SDS-CTAB,high-salt enzymolysis and kit,respectively.High-salt enzymolysis method was determined as the best method by the comparison of the genomic DNA integrality,concentration and purity,the digestion as well as PCR amplification by 18S.2.The pol gene(Genbank:AB016926.1)with good species specificity was chose to be the endogerous reference gene of Tricholoma matsutake from Nucleotide datebase,and specific primers were designed.The species specificity of the pol gene were verified by qualitative and real-time quantitative PCR Yunnan Shangri-La Tricholoma matsutake,Sichuan Ganzi Tricholoma matsutake,Jilin Yanbian Tricholoma matsutake,Russula virescens,Agaricus deliciosus,Boletus speciosus Forst,Termitornyces albuminosus,Agaricus blazei,Agrocybe aegerita,Lentinus edodes,Pleurotus eryngii,Flammulina velutipes and Pleurotus ostreatus.And the sensitivity of qualitative PCR was 8 ng,while that of real-time quantitative PCR was 32 pg.Meanwhile,the southern blot analysis result showed that the pol gene was single copy in Tricholoma matsutake genome,and the gene sequencing result indicated that there was no allelic variation of pol gene within the same species.3.The seven factors of LAMP,including Bst DNA polymerase,temperature,the ratio of outer primers to inner primers,the concentration of both outer primers and inner primers,dNTP,betaine and Mg2+ were optimized.The sensitivity of qualitative and Eva Green real-time quantitative LAMP were 0.8 ng and 16 pg by applying the optimized reaction system respectively.The LAMP-colloidal gold nucleic acid dipstick assay of pol gene was established for the first time,by optimizing the detection time and buffer solution,the sensitivity was 0.8 ng.In order to identify whether the established detection system was effective,the four deep processed products which containing the ingredient of Tricholoma matsutake on the food labels were taken as the targets,including Tricholoma matsutake biscuit,oil Tricholoma matsutake and Tricholoma matsutake sauce.After the LAMP and visual detecting,only Tricholoma matsutake biscuit was determined to contain the Tricholoma matsutake ingredient,which was the same as the result of the common PCR amplification.