Identification Of Bubali Cornu And Pheretima Using DNA Barcoding And The Optimization Of DNA Extraction Method For Animal Medicine

Objective: COI barcoding sequence was used for identification of water buffalo horn and Pheretima medicinal material in this study.We aimed to investigate the authenticity of these animal medicines in Chinese herbal medicine market and provide a powerful tool for market regulation.We also optimized the DNA extraction method for animal medicine,in order to provide technical support for the molecular identification of animal medicine in Chinese Pharmacopoeia.Methods: Firstly,we collected the original animals and commercial herbal medicine of Bubali cornu and Pheretima in the market and obtain the COI sequences,then analyzed these sequences by MEGA 6.0 and set up DNA barcoding database.Secondly,the commercial medicinal materials were identified by using this database.We based on the DNA extraction theory of SDS and assessed the effects of two elements—different EDTA concentrations(0.025 mol·L^-1?0.25mol·L^-1?0.5mol·L^-1)and whether containing Na Cl and Triton X-100 in the lysis buffer—on the quality of the DNA extracted from different kinds of animal medicine,and optimized the lysis buffer.Results: DNA was extracted successfully from all samples,most of the samples could get ideal COI sequence.The results showed that 29% of the commercial Bubali cornu medicines were yak horns,50% of Pheretima were adulterants.We optimized the lysis buffer,the results showed that the lysis buffer of 1 % SDS,0.03mol·L^-1 Tris-HCl,0.25mol·L^-1 EDTA and 0.2mol·L^-1 Na Cl had the optimum effect on DNA extraction of animal medicine.Conclusion: The COI sequences of DNA barcoding can identify Bubali cornu and Pheretima from its adulterants accurately and effectively,which provide a strong guarantee for the safety of animal medicines.The DNA extraction method established in this study can be used in the DNA extraction of different kinds of animal medicine and the obtained DNA can satisfy the requirements of molecular identification for animal medicine.This method can also lay solid foundation for the popularization and application of molecular identification in animal medicine.