平贝母特异性PCR鉴别方法研究

Study on Specific PCR Identification of Fritillaria ussuriensis

目的:建立一种客观、准确、有效的平贝母位点特异性PCR鉴别方法。方法:收集平贝母及其他贝母材料,提取样品总基因组DNA,分析比对平贝母及其他贝母ITS片段,根据差异位点设计平贝母特异性引物,采用两步法进行PCR扩增,优化反应体系并对该方法进行耐受性和适用性考察。结果:建立了平贝母的位点特异性PCR鉴别方法:引物的序列为PB-Y1F:CGGGCGGACAATTTACTT,PB-Y1R:AGATAGAGAGCGGGCGTC;反应体系:2×Taq Plus Master Mix 12.5μL,上下游引物各1μL(10μmol),DNA模板1μL,ddH_(2)O 9.5μL;扩增程序:95℃1 min;95℃10 s,57℃25 s,30个循环;72℃15 s。平贝母均能扩增出约186 bp片段,其他贝母及阴性对照无条带,方法的适用性良好。结论:该实验设计的平贝母特异性引物专属性强,所建立的特异性PCR方法可以用于平贝母药材的准确鉴别。

Objective:To establish an objective,accurate and effective site-specific PCR identification method of Fritillaria ussuriensis Methods:Fritillaria ussuriensis and other materials were collected,the total genomic DNA of the samples was extracted,and the ITS fragments of Fritillaria ussuriensis and others were analyzed and compared.Specific primers of Fritillaria ussuriensis were designed according to the different loci,and the two-step method was used for PCR amplification.The reaction system was optimized and the tolerance and applicability of the method were investigated.Results:A site-specific PCR method was established for the identification of Fritillaria ussuriensis.The primers were PB-Y1 F(CGGGCGGACAATTTACTT)and PB-Y1 R(AGATAGAGAGCGGGCGTC).The reaction system was 2×Taq Plus Master Mix 12.5μL,upstream and downstream primers each 1μL(10μmol),DNA template 1μL,ddH_(2)O 9.5μL,amplification procedure was 95℃for 1 min;95℃for 10 s;57℃for 25 s,30 cycles;72℃for 15 s.The fragment of about 186 bp was amplified from Fritillaria ussuriensis,and there was no band in others and negative control.The method has good applicability.Conclusion:The specific primers of Fritillaria ussuriensis designed in this study are highly specific,the established specific PCR method can be used for accurate identification of Fritillaria ussuriensis.