Objective: To establish the high-resolution melting(HRM) identification method for different ingredients in Tibetan patent medicine Shiliu Jianwei powder. Methods: The internal transcribed spacer Ⅱ of nuclear ribosomal DNA(ITS2) sequence from the same or similar species of the active pharmaceutical ingredient (API) which was used as the template to design primers with Primer Primer 6.0 software, according to the primer design principle. The method of agarose gel electrophoresis was used to select the specific primers that showed better amplification effect on target APIs. The HRM method was used to verify the specific primers selected by agarose gel electrophoresis, and the consistency of the detection results of the homemade Shiliu Jianwei powder and the commercially available Shiliu Jianwei powder were compared. The number of amplification cycles of the HRM experiment was optimized, the best specific primers for each API was determined, and a more specific specific HRM method was established. Results: Fifty-two pairs of specific primers were designed in the experiment, and 11 pairs of specific primers with better amplification specificity were selected by agarose gel electrophoresis detection. The HRM method was used to verify the 11 pairs of specific primers selected by agarose gel electrophoresis, the specificity of the results was consistent with the results of agarose gel electrophoresis, and the test results in the homemade Shiliu Jianwei powder and the commercially available Shiliu Jianwei powder were consistent. The best specific primers in the 5 APIs were determined. The best specific primer for Semen Punicae was SLZ-7A7S (32 cycles), the best specific primer for Carthami Flos was HH-2A2S (25 cycles), the best specific primer for Cinnamomi Cortex was RGCA-1A1S (40 cycles), the best specific primer for Amomi Fructus Rotundus was DKAC-1A1S (27 cycles), and the best specific primer for Fructus Piperis Longi was BBPBN-1A1S (35 cycles). Conclusion: Finally, the optimal amplification primers and the optimal amplification cycle number of each API in the Tibetan patent medicine Shiliu Jianwei powder for HRM detection method are determined, and the more specific identification of each API in Shiliu Jianwei powder is realized. It provides research ideas and reference for the molecular biology quality control of Chinese patent medicines with original powder.
LIU Jie, FANG Wen-liang, TANG Zhe, QIAO Fei, LIAN Chao-jie, DAI Sheng-yun, GUO Li-nong, ZHENG Jian, MA Shuang-cheng
. Based on specific primer for the HRM identification method study of Tibetan patent medicine Shiliu Jianwei powder*[J]. Chinese Journal of Pharmaceutical Analysis, 2022
, 42(8)
: 1335
-1344
.
DOI: 10.16155/j.0254-1793.2022.08.06
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