目的:基于特异性引物设计和高分辨率熔解曲线(HRM)技术建立藏成药石榴健胃散中不同药味的HRM鉴别方法。方法:以原料药相同或相近物种的ITS2序列作为模板,按照引物设计原则,利用Primer Primer 6.0软件进行引物设计;利用琼脂糖凝胶电泳方法,筛选对目标原料药扩增效果较好的特异性引物;利用HRM方法对琼脂糖凝胶电泳检测筛选出的特异性引物进行验证,比较自制石榴健胃散成药与市售石榴健胃散检测结果的一致性;优化HRM实验的扩增循环数,并确定各原料药的最佳特异性引物,建立较为专属性的特异性HRM方法。结果:实验设计了52对特异性引物,通过琼脂糖凝胶电泳检测,筛选出了具有较好扩增特异性的11对特异性引物;采用HRM方法对琼脂糖凝胶电泳检测结果筛选出的11对特异性引物进行验证,其结果的特异性与琼脂糖凝胶电泳检测结果一致,且以11对特异性引物检测自制石榴健胃散成药与市售石榴健胃散的结果一致;确定五味原料药中最佳特异性引物,石榴子为SLZ-7A7S(32 cycles),红花为HH-2A2S(25 cycles),肉桂为RGCA-1A1S(40 cycles),豆蔻为DKAC-1A1S(27 cycles),荜茇为BBPBN-1A1S(35 cycles)。结论:最终确定了石榴健胃散中各个原料药HRM检测方法的最佳扩增引物及最适扩增循环数,实现了对石榴健胃散中各个原料药较为专属性的鉴别,为原粉入药的中成药的分子生物学质量控制提供了研究思路及参考依据。
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.
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