摘要
目的:建立基于DNA微型条形码的炮山甲及其混伪品的分子鉴定技术。方法:采用改良的DNA提取方法提取中华穿山甲与其易混物种炮制甲片的DNA,利用线粒体COⅠ、16S rRNA和12S rRNA基因的标准及微型条形码引物进行PCR。双向测序后进行BLAST和序列位点分析,再计算种内和种间K2P遗传距离,并构建NJ系统树。结果:16S rRNA(L2513/H2714)和12S rRNA(L1085/H1259)引物对在所有甲片DNA中均能扩增成功,所获序列与Genbank序列的BLAST比对相似度大于98%。中华穿山甲与其易混物种的种间遗传距离远大于其种内遗传距离,而且各物种在NJ树中表现出单系性,其中16S rRNA微型条形码能将穿山甲属聚在一起。结论:基于16S rRNA的微型条形码技术可有效鉴别炮山甲及其混伪品。
Objective:To establish a molecular identification technique for the processed Manis pentadactyla and its adulterants based on DNA miniature barcodes.Methods:The genomic DNA fragments of processed Manis pentadactyla and its adulterants were extracted by optimized DNA extraction method,the standard and miniature barcode primer of mitochondrial COⅠ,16S rRNA and 12S rRNA genes were used for PCR.BLAST and sequence site analysis were performed after bidirectional sequencing,and the intraspecific and interspecific K2P genetic distances were calculated and the NJ system tree was constructed.Results:Both primer pairs including 16S rRNA(L2513/H2714)and 12S rRNA(L1085/H1259)successfully amplified the target sequences in the genomic DNAs of all processed samples,the comparison similarity between the obtained sequences and BLAST from Genbank sequences was more than 98%.The interspecific genetic distances between Manis pentadactyla and its adulterants were much larger than intraspecific genetic distances,and each species in the NJ tree showed monogamy,while 16S rRNA miniature barcodes could group the Manis genus into one group.Conclusion:The 16S rRNA miniature barcodes technology can effectively identify the Manis pentadactyla and its adulterants.
作者
陈宏
王红雨
何铭琪
杨帆
李海峰
CHEN Hong;WANG Hong-yu;HE Ming-qi;YANG Fan;LI Hai-feng(College of Life Sciences and Biopharmaceuticals,Guangdong Pharmaceutical University,Guangzhou 510006,China;Material Evidence Identification Center,Ministry of Public Security,Beijing 100038,China)
出处
《中药材》
CAS
北大核心
2019年第10期2267-2272,共6页
Journal of Chinese Medicinal Materials
基金
公安部物证鉴定中心项目(2016FGKFKT03)
广东省自然科学基金项目(2017A030313864)
关键词
中华穿山甲
炮制甲片
分子鉴定
DNA微型条形码
线粒体16S
rRNA
Manis pentadactyla Linnaeus
Processed scales
Molecular identification
DNA miniature barcodes
Mitochondrial 16S rRNA
