[Objective] The aim was to establish the optimal rye-specific PCR reaction system for rye.[Method] The ordinary wheat ＂Chinese Spring＂,S165,rye,octoploid triticale and hexaploid triticale were used as materials to c...[Objective] The aim was to establish the optimal rye-specific PCR reaction system for rye.[Method] The ordinary wheat ＂Chinese Spring＂,S165,rye,octoploid triticale and hexaploid triticale were used as materials to carry out study on the effect of the amount of template DNA,primers,dNTPs,Mg2＋ concentrations,Taq DNA polymerase and annealing temperature on the rye-specific PCR reaction system of rye.[Result] The genomic DNA extracted by modified CTAB DNA extraction method showed high quality,which was satisfied for the PCR reaction template.The rye-specific PCR reaction system was 25 μl,including 10 × buffer solution,1.5 mmol/L MgCl2,200 μmol/L dNTP,40 ng primers,40-60 ng DNA template and 1 U Taq DNA polymerase.[Conclusion] The optimal rye-specific PCR reaction system was established,which provided basis for the identification of exogenous germplasm of rye in wheat background.展开更多
目的:基于巢式聚合酶链式反应(PCR)理念设计并筛选出可用于高效扩增与鉴别紫草市场样品真伪的特异性引物。方法:针对新疆紫草的ITS序列和紫草非《中华人民共和国药典》品ITS2序列,利用Primer Premier 5软件进行巢式引物设计;比较ITS2通...目的:基于巢式聚合酶链式反应(PCR)理念设计并筛选出可用于高效扩增与鉴别紫草市场样品真伪的特异性引物。方法:针对新疆紫草的ITS序列和紫草非《中华人民共和国药典》品ITS2序列,利用Primer Premier 5软件进行巢式引物设计;比较ITS2通用引物PCR和巢式PCR对紫草药材基因组DNA的扩增效率;基于巢式引物直接扩增紫草基因组DNA,并进行琼脂糖凝胶电泳检测;基于扩增产物片段长度、变异位点覆盖情况对设计的紫草药材特异性引物进行评价。结果:经过Primer Premier 5软件进行引物设计共选出11对引物进行合成;巢式PCR对紫草药材基因组DNA的扩增效率明显优于ITS2通用引物PCR;基于巢式引物直接扩增紫草基因组DNA琼脂糖凝胶电泳检测的结果明显优于ITS2引物直接扩增紫草基因组DNA,且呈单一条带;确定AE-9S/AE-2A、AE-4S/AE-10A、AE-12S/10A、AE-29S/AE-29A共4对引物适用于紫草正伪品鉴定。结论:在DNA条形码鉴定和巢式PCR技术的基础上,确定了4对可以用于有效区分药材市场中主流的新疆紫草和紫草非《中华人民共和国药典》品的特异性引物,为后续紫草药材及其他中药民族药品种的鉴定方法研究与开发提供了可参考的依据。展开更多
基金Supported by Young Academic Backbone Support Program of Heilongjiang Province(1152G047)Student Innovation Project of Mudanjiang Normal University~~
文摘[Objective] The aim was to establish the optimal rye-specific PCR reaction system for rye.[Method] The ordinary wheat ＂Chinese Spring＂,S165,rye,octoploid triticale and hexaploid triticale were used as materials to carry out study on the effect of the amount of template DNA,primers,dNTPs,Mg2＋ concentrations,Taq DNA polymerase and annealing temperature on the rye-specific PCR reaction system of rye.[Result] The genomic DNA extracted by modified CTAB DNA extraction method showed high quality,which was satisfied for the PCR reaction template.The rye-specific PCR reaction system was 25 μl,including 10 × buffer solution,1.5 mmol/L MgCl2,200 μmol/L dNTP,40 ng primers,40-60 ng DNA template and 1 U Taq DNA polymerase.[Conclusion] The optimal rye-specific PCR reaction system was established,which provided basis for the identification of exogenous germplasm of rye in wheat background.
文摘目的:基于巢式聚合酶链式反应(PCR)理念设计并筛选出可用于高效扩增与鉴别紫草市场样品真伪的特异性引物。方法:针对新疆紫草的ITS序列和紫草非《中华人民共和国药典》品ITS2序列,利用Primer Premier 5软件进行巢式引物设计;比较ITS2通用引物PCR和巢式PCR对紫草药材基因组DNA的扩增效率;基于巢式引物直接扩增紫草基因组DNA,并进行琼脂糖凝胶电泳检测;基于扩增产物片段长度、变异位点覆盖情况对设计的紫草药材特异性引物进行评价。结果:经过Primer Premier 5软件进行引物设计共选出11对引物进行合成;巢式PCR对紫草药材基因组DNA的扩增效率明显优于ITS2通用引物PCR;基于巢式引物直接扩增紫草基因组DNA琼脂糖凝胶电泳检测的结果明显优于ITS2引物直接扩增紫草基因组DNA,且呈单一条带;确定AE-9S/AE-2A、AE-4S/AE-10A、AE-12S/10A、AE-29S/AE-29A共4对引物适用于紫草正伪品鉴定。结论:在DNA条形码鉴定和巢式PCR技术的基础上,确定了4对可以用于有效区分药材市场中主流的新疆紫草和紫草非《中华人民共和国药典》品的特异性引物,为后续紫草药材及其他中药民族药品种的鉴定方法研究与开发提供了可参考的依据。
