基于ITS序列位点特异性PCR的酸枣仁及其混伪品的鉴别

Identification of Zizyphus jujuba with the Counterfeit Products by Allele-specific PCR Based on ITS Genes Sequence

目的：基于ITS序列建立酸枣仁及其市场混伪品的分子鉴定方法。方法：收集酸枣仁及其常见混伪品理枣仁、枳椇子、紫荆子、兵豆,提取总DNA,基于ITS序列设计引物并进行PCR扩增,对扩增产物进行测序,用Bio Edit分析软件进行序列比对,针对变异位点用Primer Premier 5.0设计特异性鉴别引物,进行PCR反应并考察反应条件。结果：ITS序列可以用于酸枣仁及其易混淆品种的鉴别研究,在位点特异性PCR反应条件考察中,DNA模板用量范围应为0.7-2.1 ng·μL^-1,退火温度在59℃时特异性最佳,实验建立的方法对不同Taq酶和PCR仪具有普遍适应性。结论：研究设计的位点特异性引物在一定条件下的PCR反应中,酸枣仁能扩增出66 bp的条带,而其他混伪品不能扩增出条带,从而实现了酸枣仁与其混伪品的快速、准确鉴别。

Objective： To establish molecular identification method for Zizyphus jujuba and its counterfeit species based on ITS genes sequence. Methods： Total DNAs of Z. jujuba,Z. mauritiana,Hovenia dulcis,Lens culinaris and Cercis chinensis were extracted, and the internal transcribed spacer（ ITS） regions were amplified by PCR and the products were sequenced. Bio Edit analysis software was used for sequence comparison. Primer Premier 5. 0 was applied to design specific primers according to the mutation sites,the PCR reactions were performed and the conditions were examined. Results： ITS sequences were used in identification research of Z. jujuba and its counterfeit species. To investigate the specific PCR reaction conditions,the scope of DNA template should variate from 0. 7-2. 1 ng·μL^-1,the annealing temperature was proper at 59 ℃,different Taq enzymes and PCR instruments were available for this PCR process. Conclusion： In the same PCR reaction,66 bp DNA band could be amplified from Z. jujuba while nothing from the counterfeit species. It is a fast and accurate method to identify Z. jujuba from the counterfeit products using specific PCR primer.