目的 研制一种集人参DNA提取与聚合酶链式反应(PCR)鉴定于一体的快速DNA检测试剂盒。方法 对经典人参DNA提取及PCR鉴定方法进行改良,确定试剂盒的组成与反应条件,并评价其性能指标。用试剂盒对人参正品、伪品进行DNA提取和PCR扩增,紫外分光光度法测纯度,并对市售人参样品进行验证。结果 提取的 DNA 纯度为(1.73±0.13);正品人参PCR鉴定结果在150~200 bp处有单一明亮条带。试剂盒特异性为100%;批内、批间变异系数分别为2.38%和2.62%;样品DNA原液稀释200倍仍可检测;反复冻融 20 次对检测效果无影响,可于-20 ℃保存 1 年;10个市售样品中8个为正品,2个为伪品。结论 人参 DNA 检测试剂盒提取的核酸浓度与纯度满足鉴定需求,且特异性好、灵敏度高、稳定性强,适用于人参及其伪品的快速检测。
Abstract
OBJECTIVE To develop a rapid DNA detection kit for DNA extraction and PCR identification of Panax ginseng C.A. Mey. METHODS The classical DNA extraction and PCR identification METHODS for Panax ginseng C.A. Mey were modified, and the compositions and reaction conditions of the kit were determined. In addition, the specificity, stability, sensitivity, and repeatability of the kit were evaluated. The genomic DNAs of genuine and counterfeit ginseng goods were extracted by the kit and PCR was performed to identify the authenticity. The purity of the extracted DNA was detected by UV spectrophotometry. Finally, commercially available ginseng samples were verified. RESULTS The purity of the genomic DNA extracted by the kit was (1.73±0.13)(OD260/ OD280), and a fragment between 150 and 200 bp could be amplified only from authentic Panax ginseng C.A. Mey. The specificity of the kit was 100%. The repetitive experiments showed that the average intra-assay CV% and inter-assay CV% of the kit were 2.38% and 2.62%, respectively. The DNA in solutions diluted by 200 times could still be detected. Stability experiment proved that repeated freeze-thawing for 20 times had no significant effect on the activity of this kit and the test sample could be stored at -20 ℃ for one year. The specificity test confirmed that 8 samples among the 10 commercial products were genuine, and 2 were counterfeit. CONCLUSION The nucleic acid extraction and purity of the DNA detection kit can meet the requirement for identification of Panax ginseng C.A. Mey.The kit has good specificity, high sensitivity, and good stability, so it is suitable for the rapid detection of Panax ginseng C.A. Mey.
关键词
人参 /
DNA提取 /
聚合酶链式反应 /
DNA检测试剂盒 /
性能评价
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Key words
Panax ginseng C.A. Mey /
DNA extraction /
PCR identification /
DNA detection kit /
performance evaluation
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中图分类号：
R284
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参考文献
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脚注
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基金
吉林省教育厅科学技术研究资助项目(2012133);吉林省科技发展计划资助项目(20140307008YY);吉林省战略性新兴产业和高新技术产业发展资助项目(2013G030);吉林市科技计划资助项目(2015420013)
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