中药穿山甲DNA分子鉴定研究.docVIP

中药穿山甲的DNA分子鉴定研究 　　[摘要] 对中药穿山甲及其混伪品进行分子鉴定研究，建立一个稳定、准确的位点特异性PCR鉴别体系。收集整理穿山甲属动物组织样本及NCBI序列，提取基因组总DNA，PCR扩增Cytb和COⅠ序列并测序，通过BioEdit软件进行序列比对，应用MEGA 6.0构建序列系统聚类树，并根据序列特异位点设计和筛选中华穿山甲特异性鉴别引物，并对PCR反应条件进行退火温度、DNA模板上样量和PCR循环数等条件的适应性考查。结果表明：Cytb和COⅠ序列均能有效对穿山甲正、伪品进行聚类分析；在位点特异性PCR反应体系中，当退火温度为55～60 ℃，DNA模板量3～100 ng，PCR扩增35个循环时，基于COⅠ序列的引物COⅠ-S10/A5 能使中?A穿山甲扩增出约400 bp的特异性条带，而其他穿山甲品种扩增结果为阴性。该文基于COⅠ序列的引物COⅠ-S10/A5能实现中华穿山甲与伪品印度穿山甲、马来穿山甲、树穿山甲等的准确、稳定鉴别。 　　[关键词] 穿山甲；COⅠ；Cytb；分子鉴定 　　[Abstract] The study was aimed to establish a stable，accurate site specific PCR identification system to identify Manis pentadactyla and its adulterants using DNA molecular identification. The genomic DNA was extracted from experimental samples using the DNA extraction kit. The Cytb and CO Ⅰ genes were amplified using PCR and sequenced bi-directionally. Obtained sequences were assembled using the BioEdit software. The neighbor-joining tree was constructed by MEGA 6.0. Specific identification primers were designed according to the specific allelets，and PCR reaction system was optimized. The results indicated that the Cytb and CO Ⅰ sequence both were able to be used to identify M. pentadactyla and its adulterants. With the specific primers CO Ⅰ-S10/A5，the M. pentadactyla could be amplified a 400 bp DNA band when the annealing temperature ranged from 55 to 60 ℃ and the amount of DNA template ranged from 3 to 100 ng within 35 PCR cycles. However，other adulterants displayed no relevant bands. So that primers CO Ⅰ- S10 / A5 can be used to identify the M. pentadactyla with the adulterants. 　　[Key words] Manis pentadactyla；COⅠ；Cytb；molecular identification 　　穿山甲为鲮鲤科Manidae穿山甲属Mains动物，全球现存包括分布亚洲的中华穿山甲M. pentadactyla、印度穿山甲M. crassicaudata、马来穿山甲M. javanica、菲律宾穿山甲M.culionensis 4种，分布非洲的树穿山甲M. tricuspis、大穿山甲M. gigantea南非穿山甲M.temminckii和长尾穿山甲 M. tetradactyla 4种[1]。其中中华穿山甲是我国药典规定的中药穿山甲的唯一正品来源，是我国传统名贵中药之一[3-6]，具有活血消?Y、通经下乳、消肿排脓、搜风通络之功效，用于经闭症瘕、乳汁不通、痈肿疮毒、风湿痹痛、中风瘫痪、麻木拘挛等症。然而近年来中华穿山甲的资源极度稀缺，早在2013年世界自然保护联盟（IUCN） 物种生存委员会（SSC） 穿