Study On DNA Barcoding Screening Of Toxic Mushroom With Liver And Kidney Damage

The poisonous mushrooms that cause acute liver damage and acute renal failure mainly belong to 4 groups,Amanita,Cortinarius,Galerina,and Lepiota.Both of these two types of poisoning have a certain incubation period,and it is necessary to take timely treatment based on the symptoms of poisoning and the cause of poisoning after the onset of the disease,but it is limited due to the existence of closely related species and similar species,and it is often difficult to collect complete samples after the poisoning event occurs,and may need to obtain the samples from leftovers,cooked mushrooms,vomitus,or gastric extracts for identification,which all make effectively identifying poisonous mushrooms more difficult.Therefore,it is necessary to find a more rapid and accurate identification method,and DNA barcoding technology is a better solution at present.In this study,60 materials of 23 species of genus Amanita,60 materials of 28 species of genus Cortinarius,40 materials of 16 species of genus Galerina,27 materials of 16 species of genus Lepiota were used as the test specimens.ITS,IGS,nr LSU,mt SSU,RPB1,RPB2,EF1-α,β-tubulin were selected as candidate genes.Amplification and sequencing were conducted using universal eukaryotic primers.Through the materials and candidate genes above,the DNA barcodes of 4genera were screened based on the three evaluation indicators,including intra-and inter-specific pairwise distance,the difficulty of sequence acquisition(sequence acquisition rate),and the ability of distinguishing species.The results showed that among the four genera,the ITS sequences had large intra-and inter-specific pairwise distance and were relatively conserved within the species,with obvious Barcoding Gap,and the sequences were easy to amplify and sequence,and could accurately distinguish the species within the four genera.EF1-α and β-tubulin sequences have high versatility only in the genus Amanita,easy to amplify and sequence,with large variation between species and relatively conservative within species,and the Barcoding Gap is obvious,so it is recommended to use ITS,EF1-α and β-tubulin gene as ideal DNA barcode of genus Amanita,and different gene fragments can be used to amplify according to actual needs in the application process to achieve the best effect.The ITS gene is suitable as ideal DNA barcode for the genus Cortinarius,Galerina and Lepiota.On this basis,this study also developed DNA mini-barcode sequences and designed primers of the four genera,and verified and analyzed the versatility and applicability of the primers.The results showed that these primers had good versatility,and strong applicability with low requirements on material quality,and amplifying materials that DNA was degraded due to cooking or digestion,which has certain practical application significance.Furthermore,this study developed the nucleotide signature sequences of two species,Cortinarius tubarius and Galerina calyptrata,and designed corresponding specific primers.In the process of species identification,the sequencing link can be omitted,and agarose gels can be used.Species can be accurately identified by observing bands of electrophoresis,which provides a new research direction for the molecular identification method of poisonous mushroom species.