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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Bio Edit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能使中华穿山甲扩增出约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。
On the Chinese medicine pangolin and its adulterants molecular identification studies to establish a stable and accurate site-specific PCR identification system. The samples of Pangolinia were collected and sequenced, the total genomic DNA was extracted, the Cytb and COⅠ sequences were amplified by PCR and sequenced. The sequences were aligned by Bio Edit software. The phylogenetic tree was constructed by MEGA 6.0. Design and screening of Chinese pangolin-specific primers, and PCR reaction conditions for annealing temperature, DNA template loading and PCR cycles and other conditions of the fitness test. The results showed that both Cytb and COⅠ sequences could effectively cluster Pangolin positive and false; in the site-specific PCR reaction system, when the annealing temperature was 55 ~ 60 ℃, the amount of DNA template was 3 ~ 100 ng, At 35 cycles, the COⅠ-sequence-based primer COⅠ-S10 / A5 amplified about 400 bp in the pangolin, while the other Pangolin species did not amplify. The COⅠ-sequence-based primer COⅠ-S10 / A5 can accurately and stably identify the Chinese pangolins and the counterfeit Indian pangolins, pangolin and pangolin.