目的: 基于酶促恒温扩增(enzymatic recombinase amplification, ERA)技术建立一种可快速定性鉴别道地药材天麻真伪的方法。方法: 遵循ERA引物设计原则,应用Oligo 7.0软件根据天麻及其常见伪品的ITS2基因组序列,筛选优化天麻ERA特异性引物,应用Primer Premier 5.0软件设计天麻特异性PCR鉴别引物,通过对ERA、PCR反应体系的优化,最终确定ERA最佳反应时间为17 min,最适反应温度为40 ℃;PCR最优退火温度为57 ℃,循环为32次,对所建立的方法进行灵敏度、特异性验证,并选取中药市场市售天麻样品进行检测。结果: 所设计的天麻ERA鉴别引物与其常见伪品间无交叉反应,特异性良好;重复性结果显示,3次重复性检测结果一致,未出现假阳性与假阴性;该方法对天麻基因组DNA的灵敏度为1 pg·μL-1,比传统PCR灵敏性高;ERA技术能够用于天麻市售样品的快速鉴定,且检测结果与PCR方法相同。结论: 所建立的检测方法操作简单、快速,具有较高的特异性和灵敏度,为道地药材天麻的真伪鉴别提供了一种新手段。
Objective: To establish a method for the rapid identification of the authenticity of Gastrodiae Rhizoma herbs based on enzymatic recombinase amplification (ERA) technique. Methods: Following the principle of ERA primer design, Oligo 7.0 software was applied to screen and optimize the ERA-specific primers of Tianma based on the ITS2 genome sequences of Gastrodia elata and its common artifacts. Primer Premier 5.0 software was applied to design the specific PCR primers for the identification of Gastrodia elata, and through the optimization of ERA and PCR reaction system, the optimal reaction time for ERA was finally determined to be 17 min, the optimal reaction temperature was 40 ℃, the optimal annealing temperature for PCR was 57 ℃, and the cycle was 32 times, and the established method was verified for sensitivity and specificity, and the samples of asparagus available in the traditional Chinese medicine The sensitivity and specificity of the established method were verified, and the commercially available asparagus samples in the market were selected for testing. Results: The designed primers for the specific identification of ERA in Gastrodia elata did not cross-react with its common forgeries, and the specificity was good, a repeatability results showed that the three repeatability tests were consistent, with no false positives or false negatives; the sensitivity of this method for Gastrodia elata genomic DNA was 1 pg·μL-1, which was higher than that of conventional PCR; the ERA technique can be used for the rapid identification of commercially available samples of Gastrodia elata and the results of the assay are the same as those of the PCR method. Conclusion: The established detection method is simple, rapid, with high specificity and sensitivity, and provides a new means for the authentication of the Gastrodiae Rhizoma.
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