目的: 建立针对真菌类中药材茯苓的聚合酶链式反应(polymerase chain reaction, PCR)鉴定技术,从分子生物学角度出发,对茯苓的真伪进行鉴定。方法: 对经典十六烷基三甲基溴化铵(CTAB)法进行改良,提取样品基因组DNA,以茯苓内转录间隔区(internal transcribed space,ITS)为目的序列,设计特异性引物,优化PCR反应体系,建立茯苓PCR鉴定方法,同时克隆特异性扩增的目的条带,测序鉴定其准确性,最后进行方法学验证。结果: 提取的基因组DNA浓度较高、纯度较好、完整度良好。琼脂糖凝胶电泳结果表明正品茯苓可以在199 bp处出现单一明亮的特异性扩增条带,而伪品不出现扩增条带,引物的特异性为100%,克隆后的目的片段电泳图谱清晰明亮,克隆片段与Genbank数据库中已登记的茯苓物种序列相似性高达100%。在操作人员不知样品真伪的情况对样本检测,目标条带出现位置与预期一致;灵敏度检测结果表明,DNA检测下限可达1 ng·μL-1;挑选3位实验室人员进行茯苓真伪检测,结果相同且和预期一致,建立的茯苓PCR鉴定方法特异性良好,准确性高,重复性好。结论: PCR鉴定技术为茯苓的真伪鉴别提供了简便可靠、特异性良好,并且准确性高的鉴别方法。采用分子克隆技术可以成功保存茯苓标准对照品,测序结果准确可靠。
Objective: To establish PCR identification technology of Poria cocos (Schw.) Wolf, a Chinese medicinal material of the fungus, and identify the authenticity of Poria cocos (Schw.) Wolf from the perspective of molecular biology. Methods: The classic CTAB method was improved and used to extract the genomic DNA from the samples, specific primers were designed with the internal transcription spacer of Poria cocos (Schw.) Wolf as the target sequence, and PCR reaction system was optimized. PCR identification method of Poria cocos (Schw.) Wolf was established. At the same time, the target band specifically amplified was cloned and sequenced to identify its accuracy. Finally, the method was verification. Results: The extracted genomic DNA was of high concentration, good purity, and good integrity. The results of agarose gel electrophoresis showed that the genuine Poria cocos (Schw.) Wolf could appear a single bright specific amplified band at 199 bp, but the counterfeit could not appear amplified band. The specificity of the primer was 100%. It was cloned and the target fragment was clear and bright after the electrophoresis pattern. And the cloned fragment had 100% similarity with sequence of Poria cocos (Schw.) Wolf registered in Genbank database. When the operator didn’t know the authenticity of the samples, the samples were tested, and the position of the target bands were consistent with the expectation. The sensitivity test results showed that the lower limit of DNA detection was up to 1 ng·μL-1. Three laboratory staff were selected for testing the authenticity of Poria cocos (Schw.) Wolf, and the results were the same and consistent with expectations. The established PCR identification method showed good specificity, high accuracy and repeatability. Conclusion: PCR identification technology provides a simple, reliable, specific and highly accurate identification method for the authenticity of Poria cocos (Schw.) Wolf. Its standard reference materials can be successfully stored by using molecular cloning technology and the sequencing results are accurate and reliable.
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