【摘要】 目的 通过聚合酶链式反应（PCR）与层析试纸条结合的方法，实现天麻快速可视化真伪鉴别，并对其检测效果进行评价。方法 采用一步法提取天麻及其伪品基因组DNA，应用NCBI数据库设计天麻特异性引物。采用DNA分子克隆技术制备天麻阳性质粒，作为天麻阳性对照品。建立PCR-层析试纸条法鉴定天麻真伪，摸索最优实验条件，并进行方法学评价。结果 (1)样品DNA提取的纯度符合要求，最优的PCR反应引物浓度为1μmol/L，循环为29次。(2)分子克隆的天麻阳性质粒序列与天麻DNA分子标记特异性指纹区片段序列同源性为98%，可作为天麻PCR-层析试纸条法的阳性对照品。(3)PCR-层析试纸条法的方法学评价结果显示：天麻对照药材在试纸条上出现两个条带，伪品和阴性对照品出现1个条带，与琼脂糖凝胶电泳法结果一致，特异性良好；PCR-层析试纸条法较琼脂糖凝胶电泳法的灵敏度高100倍，天麻DNA浓度为10-1 mg/L时，试纸条仍有模糊条带；在第3、6、9、12个月采用PCR-层析试纸条法进行检测，检测结果与预期一致，稳定性良好；混合样品验证显示，PCR-层析试纸条法的最低检测限为10%，而琼脂糖凝胶电泳法的最低检测限为50%。(4)采用PCR-层析试纸条法对15份市售天麻样品进行检测，鉴别出3个伪品，与琼脂糖凝胶电泳法结果一致。结论 所构建的PCR-层析试纸条检测方法特异性强、灵敏度高、操作简便快速，可在短时间内实现鉴定结果的可视化，检测结果准确、稳定，为道地药材天麻的真伪鉴别提供了新方法。更多还原

【Abstract】 Objective The rapid visual identification of Gastrodiae Rhizoma was realized by the combination of polymerase chain reaction(PCR) and chromatographic test strips, and the detection effect was evaluated. Methods The genomic DNA of Gastrodiae Rhizoma and its counterfeits was extracted by one-step method, and the specific primers of Gastrodiae Rhizoma were designed using NCBI database. The positive plasmid of Gastrodiae Rhizoma was prepared by DNA molecular cloning technology as the positive control of Gastrodiae Rhizoma. The PCR-chromatography test strip method was established to identify the authenticity of Gastrodiae Rhizoma, the optimal experimental conditions were explored, and the methodology was evaluated. Results(1)The purity of DNA extracted from the samples met the requirements. The optimal PCR primer concentration was 1 μmol/L and the cycle was 29 times.(2)The homology between the positive plasmid sequence of the molecularly cloned Gastrodiae Rhizoma and the sequence of the specific fingerprint region of the DNA molecular marker of Gastrodiae Rhizoma was 98%, which could be used as a positive control for the PCR-chromatographic test strip method of Gastrodiae Rhizoma.(3)The results of PCR-chromatographic test strip methodological evaluation showed that there were 2 bands on the test strip for the Gastrodiae Rhizoma reference medicinal material, and 1 band for the counterfeit and negative control, which was consistent with the results of agarose gel electrophoresis method and with good specificity. The sensitivity of PCR-chromatographic test strip method was 100 times higher than that of agarose gel electrophoresis method. When the concentration of Gastrodiae Rhizoma DNA was 10-1 mg/L, the test strip still had fuzzy bands. At the 3 rd, 6 th, 9 th and 12 th month, the PCR-chromatography test strip method was used for detection, the test results were consistent with the expectations, and the stability was good. The validation of mixed samples showed that the minimum detection limit of PCRchromatography test strip method was 10%, while the minimum detection limit of agarose gel electrophoresis method was 50%.(4) The PCRchromatography test strip method was used to detect 15 commercial Gastrodiae Rhizoma samples, and 3 counterfeit products were identified, which were consistent with the results of agarose gel electrophoresis method. Conclusion The PCR-chromatographic test strip detection method has strong specificity, high sensitivity, simple and rapid operation, can realize the visualization of identification results in a short time, and the detection results are accurate and stable, which provides a new method for the authenticity identification of Gastrodiae Rhizoma genuine medicinal materials.更多还原