目的:建立一种能够快速鉴别中国真龙虱及其混伪品的特异性聚合酶链式反应(PCR)方法.方法:收集中国真龙虱及其混伪品共 15 批次,提取各样品基因组DNA,测序得到中国真龙虱及其混伪品的COⅠ序列,经比对后筛选出特异性单核苷酸多态性(SNP)变异位点,设计特异性鉴别引物,同时考察影响PCR反应的各项因素.结果:双向测序分别获得中国真龙虱、中华龙虱、达牙甲的COⅠ片段;中国真龙虱特异性引物在 296 bp处能扩增出单一清晰的目标条带,而在同样条件下,其混伪品均无此条带出现.结论:该研究所建立的中国真龙虱特异性PCR方法可快速而准确地鉴定中国真龙虱及其混伪品,为后续中国真龙虱药材的质量控制研究奠定了实验基础.
Objective:To establish a specific polymerase chain reaction(PCR)method for rapid identification of Cybister chinensis and its adulterants.Methods:A total of 15 batches of Cybister chinensis and its adulterants were collected,genomic DNA was extracted from all samples,and the COⅠ sequences of Cybister chinensis and its adulterants were sequenced.After comparison,the specific single nucleotide polymorphism(SNP)mutation sites were screened,specific identification primers were designed,and the factors affecting PCR reaction were investigated.Results:The COⅠ fragments of Cybister chinensis,Dytister sinensis,Hydrophilus dauricus were obtained by bidirectional sequencing.The specific primers of Cybister chinensis could amplify a single clear target band at 296 bp,but no such band appeared in adulterants under the same conditions.Conclusion:The specific PCR method established by the institute can quickly and accurately identify Cybister chinensis and its adulterants,which lays the experimental foundation for the subsequent quality control research of Cybister chinensis.
蔡庆群;谢纯珠;冯时茵;廖娴;杨棣华
广州中医药大学第一附属医院,广东 广州 510405
中医学
中国真龙虱;COⅠ序列;特异性PCR;分子鉴定
Cybister chinensis Motschulsky;COⅠ sequence;Specific PCR;Molecular identification
《中药材》 2023 (012)
2964-2968 / 5
广东省中医药局中医药科研项目(20221138)
10.13863/j.issn1001-4454.2023.12.009
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