多基原九里香药材的多重位点特异性PCR鉴别

Identification of Murrayae Folium et Cacumen by Multiplex Allele-Specific PCR

目的:建立一种同时鉴别九里香药材两基原九里香Murraya exotica和千里香M.paniculata的DNA分子鉴别方法。方法:通过比较九里香、千里香的叶绿体基因组序列差异,根据叶绿体基因组上的单核苷酸多态性(SNP)变异位点分别设计九里香和千里香的特异性鉴别引物P03和P04,对影响聚合酶链式反应(PCR)结果的退火温度、循环次数、引物浓度比进行优化,建立了九里香和千里香的多重位点特异性PCR鉴别方法,并对影响该方法重复性的Taq酶种类、PCR仪型号进行耐受性考察和适用性考察。结果:在该多重位点特异性PCR鉴别方法中,当PCR反应循环数为31次,退火温度为60℃,九里香P03和千里香P04引物比为1∶2时,九里香可获得约330 bp的特异性条带,千里香可得到约230 bp的特异性条带,不同来源的九里香和千里香样品使用建立的多重位点特异性PCR鉴别方法均可获得一致结果。结论:该文建立一种快速、准确鉴别九里香药材基原的多重位点特异性PCR鉴别方法,可在单次鉴定操作中根据条带位置同时鉴别九里香和千里香。

Objective:To establish a polymerase chain reaction(PCR) method to accurately discriminate the crude materials of Murrayae Folium et Cacumen,Murraya exotica and M.paniculata.Method:Based on the difference in chloroplast genome sequences of M.exotica and M.paniculata,species-specific identification primers P03 and P04 of M.exotica and M.paniculata were designed according to single nucleotide polymorphism(SNP)on the chloroplast genome.A multiplex allele-specific PCR identification method was established for the identification of M.exotica and M.paniculata following the optimization of annealing temperature,number of cycles,and primer concentration ratio.The established PCR method for identification was explored and verified in terms of tolerance and feasibility by investigating the type of Taq polymerases and PCR system model.Result:In this multiplex allele-specific PCR identification method,about 330 and 230 bp of specific fragments were amplified from DNA templates of M.exotica and M.paniculata,respectively,under the following conditions:cycle number of 31,annealing temperature of 60 ℃,and primer concentration ratio of P03 and P04 of 1∶2.Consistent results were obtained for samples from different sources.Conclusion:The multiplex allele-specific PCR identification method established in this study can accurately identify the origin of Murrayae Folium et Cacumen,which can be used for the simultaneous identification of M.exotica and M.paniculata by the length of fragments in a single identification assay.