火术微粉原料药火炭母与麸炒白术饮片的DNA条形码分子鉴定OA

The objective was to establish a method for extracting genomic DNA from raw medicinal materials of ultramicro-pulverised powder of Polygonum chinense L.and Atractylodes rhizome(PAUP),namely P.chinense decoction pieces and stir-fried Atractylodes macrocephala Koidz.with bran decoction pieces,and to identify the authenticity of these raw materials using DNA barcoding technology,thereby ensuring the accuracy of the original species of Chinese medicinal raw materials.Genomic DNA was extracted from the decoction pieces using the conventional CTAB method,kit method,and modified CTAB method.The internal transcribed spacer 2(ITS2)sequences were amplified by PCR,followed by sequencing,sequence assembly,annotation,and BLAST analysis for species identification of 21 commercially available P.chinense decoction pieces and 20 stir-fried A.macrocephala with bran decoction pieces,which were from different producing areas and batches.ITS2 sequences of 5 common adulterants each for P.chinense and A.macrocephala were downloaded from GenBank.MEGA 11.0 software was used to calculate the intra-specific and inter-specific K2P genetic distances of P.chinense,A.macrocephala,and their respective adulterants,and a Neighbor-Joining(NJ)phylogenetic tree was constructed to verify the effectiveness of ITS2 barcode identification.The results showed that the modified CTAB method yielded the optimal DNA purity(OD260 nm/OD280 nm=1.81)and concentration(30.84 ng/µL),with a PCR amplification success rate and sequencing success rate of 100%.BLAST analysis showed that all 20 samples of A.macrocephala decoction pieces were identified as A.macrocephala,among the 21 samples of P.chinense,1 sample was highly matched with Pueraria montana var.lobata,and the rest were identified as P.chinense.K2P genetic distance and phylogenetic tree analysis revealed that the intra-specific variation of ITS2 in both P.chinense and stir-fried A.macrocephala was small.In the NJ phylogenetic tree,P.chinense,A.macrocephala and their respective adulterants could be clustered into separate clades.The modified CTAB method yields high-quality genomic DNA from processed decoction pieces.Combined with DNA barcoding technology,it can effectively and accurately identify P.chinense and stir-fried A.macrocephala with bran decoction pieces from their common adulterants,providing strong technical support for the identification of raw medicinal materials of PAUP.