Molecular Identification Of Processed And Digested Mushroom

Poisoning from the consumption of poisonous mushrooms occurs in China,and they are difficult to identify by traditional morphological methods after they have entered the consumption and digestion chain.In recent years,DNA molecular marker technology has developed rapidly in the identification of fungal species,and DNA barcoding is a method for rapid and accurate identification of species using a segment or several short DNA sequences for rapid and accurate identification of species.Based on DNA barcoding technology,this study established a standardized operation procedure for DNA barcode identification by optimizing the PCR reaction conditions.To explore the effect of processing and digestion on mushroom DNA extraction,improve the DNA extraction method and establish a melting curve analysis detection method based on RT-PCR technology to lay the foundation for the identification of poisonous mushroom species.1.The establishment of standardized operational procedures for DNA barcode identification:A standardized procedure for DNA barcode identification was constructed by collecting wild mushrooms with optimized PCR reaction conditions.（1）A single-factor optimization study was conducted to influence the PCR amplification annealing temperature,and finally the primers ITS and LSU amplification program was established as 94℃for 5 min,94℃for 30 s,58℃for 40 s,72℃for 1 min,27 cycles,72℃for 10 min;RPB2 amplification program was 94℃for 5 min,94℃for 30 s,58℃for 30s,72℃for 1 min,24 cycles,72℃for 10 min.（2）A multiple-gradient optimization study of the factors affecting the PCR reactions of the two primers was conducted,and the optimal PCR amplification reaction conditions of Mg²⁺concentration of 1.5-2.0 mmol/L,d NTP concentration of 0.2 mmol/L,Taq enzyme dosage of 0.75-1.0 U,and DNA template dosage of 50-60 ng were derived.（3）Combining ITS+LSU+RPB2 sequence identification and morphological observation,the species identification of 30 wild mushroom samples collected showed that30 wild mushrooms belonged to 13 families,16 genera and 24 species,among which there were 13 edible mushrooms,6 medicinal mushrooms,7 species containing toxicity and 7species with unknown food toxicity.The PCR amplification success rates of the three DNA barcodes were 100%,90%,and 70%,and the sequencing success rates were 100%,90%,and 53%,respectively.Combined with the phylogenetic tree clustering analysis,their identification efficiency was ITS>LSU>RPB2 from high to low,and the combined use of the three could increase the species identification success rate to 100%.Therefore,in the established DNA barcode identification procedure,the ITS sequence can be used as the core barcode,and LSU and RPB2 as the auxiliary barcodes.2.Effect of extraction method and processing and digestion on the quality of mushroom DNA:（1）The CTAB method was improved in terms of grinding method and removal of impurities,and the experimental results showed that the homogenizer grinding method extracted DNA solutions with OD260/OD280 values of 1.97-2.0 and concentrations of555.67-1219.33μg/m L,which met the requirements of subsequent molecular experiments,and the homogenizer grinding method could both extract high-quality DNA and the homogenizer grinding method can both extract high quality DNA and save cost for easy use,so this method was chosen to grind the samples for the experiment.The removal of impurities during DNA extraction was performed in the following ways:proteinase K andβ-mercaptoethanol were added to the CTAB extract to remove proteins and polyphenols;RNase A was added after the first extraction by phenol/chloroform iso/pentanol solution to remove not only RNA,but also RNase A,eliminating the effect of RNA enzymes on DNA.The CTAB method was modified to increase the OD260/OD280 values from 1.75-2.31 to1.97-2.0 and the concentrations from 559-1131.68μg/m L to 877.33-1219.33μg/m L.PVP method and CTAB method to extract high sugar wild mushroom DNA concentration of both there is a significant difference P<0.05,PVP method can reduce to improve the difficulty of high sugar wild mushroom DNA extraction success rate,the removal of polysaccharides has a significant effect.（2）The DNA of the processed and digested samples was extracted by the kit method and the modified CTAB method,and the experimental results showed that the DNA concentration of the extracted samples decreased gradually with the prolongation of the boiling time of the boiled samples.High concentrations of DNA could be extracted from the dried samples with concentrations of 22.33-1187.33μg/m L.The oil content of the fried samples was high thus affecting the extraction of DNA.The fried samples were de-oiled using methanol/chloroform solution and the de-oiling treatment improved the DNA quality up to a concentration of 1339.33μg/m L.The kit method failed to extract DNA from individual digested samples,while the modified CTAB method could extract DNA from all samples,and the extracted DNA was more complete.The average concentration of DNA extracted from processed and digested samples reached 1291.05μg/m L by the modified CTAB method and 89.53μg/m L by the kit method.Compared with the kit method,the modified CTAB method was more operable,the extracted DNA concentration was higher,and the OD values were more stable,all in the range of 1.8-2.0.（3）The DNA extraction method and PCR reaction procedure established were validated using the wild poisonous mushroom Coprinopsis atramentaria,and the modified CTAB method was suitable for extracting DNA from high concentration and high quality processed digested samples,and the resulting samples had OD260/OD280 values between 1.8-2.0 and DNA concentrations of 408.67-1248.67μg/m L,and the success rates of primer ITS and LSU amplification were 100%.3.Establishment of a high-resolution melting based RT-PCR identification method:By using the high-resolution melting of RT-PCR,the RT-PCR primer COA1 was designed according to the ITS sequence of 12 samples,and the rapid detection method of fluorescent PCR was established by analyzing the melting Tm value and melting peaks to identify different mushroom species.Results showed that the designed primer COA1 was suitable for RT-PCR identification and could distinguish 12 mushrooms with no overlap of melting peaks,and all samples had Ct values less than 35 and no non-specific amplification and primer dimerization.The method can be used for the rapid identification of toxic mushrooms without the conventional sequencing steps.