鹿胎阳性质控品DNA检测试剂盒的开发与应用

目的 研发鹿胎DNA阳性质控品,组装检测试剂盒.方法 提取鹿胎及其伪品DNA,设计通用鹿引物并进行PCR扩增,克隆种属特异性目的片段,构建鹿胎DNA阳性质控品并组装DNA检测试剂盒,进行试剂盒性能实验并应用于市售鹿胎样品检测.结果 应用美国国家生物技术信息中心(NCBI)分析测序比对结果,正确率为100％,在526 b...     

梅花鹿鞭与伪品牛鞭细胞色素b DNA指纹鉴定

目的 探讨梅花鹿鞭及牛鞭线粒体细胞色素b(cytochrome b,Cytb)基因特异性片段特征,建立梅花鹿鞭的DNA指纹鉴定方法.方法 采用引物设计软件Primer Premier 5.0设计了用于鉴定梅花鹿鞭的特异性引物,用此引物对市售的梅花鹿鞭样本进行DNA指纹鉴定.结果 梅花鹿鞭能扩增出306 bp大小的片段,而牛鞭未能扩增出相应片段.结论 梅花鹿鞭DNA具有特异性指纹特征,所得梅花鹿鞭DNA指纹特征图谱可用于市售梅花鹿鞭的鉴定.     

基于PCR技术可视化鉴别金钱白花蛇方法的建立

目的 基于聚合酶链式反应(PCR)技术,利用核酸扩增产物,建立一种可以快速鉴定金钱白花蛇中药材真伪的可视化试纸条检测方法。方法 提取金钱白花蛇、赤链蛇及市售样本的基因;根据mtDNA Cytb序列进行对比分析,设计1对特异性鉴别引物,进行修饰物标记;建立鉴别PCR反应体系,使用核酸扩增产物,利用可视化试纸条对市售样本进行真伪鉴定。结果 鉴别引物可将正品金钱白花蛇中药材扩增出500~600 bp之间的特异性条带,而伪混品则不出现相应的特异性条带;可视化试纸条对市售样本进行检测,鉴别效果良好,灵敏度高,可准确鉴定中药材真伪。结论 可视化试纸条可以快速鉴别金钱白花蛇中药材真伪,稳定性高、特异性强,可应用于市场出售金钱白花蛇中药材的鉴定。     

中药材鹿茸的分子分类学鉴定研究

目的 建立中药材鹿茸的分子分类学鉴定试剂盒。方法 根据对不同产地的梅花鹿、马鹿线粒体DAN进行PCR扩增和序列测定，并与亲缘关系较近的伪充药材来源动物线粒体DNA同位置序列比较，找到鹿的特征片段，经过实际经验，筛选出适合片段作为鹿的种属特异性PCR引物。结果 该引物与相关试剂组成试剂盒后，可用于中药材鹿茸与伪充药材的鉴别。结论 用分子分类学方法鉴别中药材鹿茸，具有科学、稳定、准确、简便等特点，值得推广。     

鹿源类中药材DNA序列分析及马鹿和梅花鹿的PCR鉴定

目的采用特异引物PCR扩增鉴定马鹿和梅花鹿源中药材。方法从马鹿、梅花鹿等国内11种鹿的鹿血和鹿茸中提取DNA,用通用引物L1091和H1478调取细胞线粒体12SrRNA基因片段,在该片段核苷酸序列比对的基础上,设计马鹿和梅花鹿高特异性PCR鉴定引物对,并进行PCR特异性鉴定。结果12SrRNA基因片段能较好地在种的水平上将不同的种区分开来;特异引物对(EP-1/H1478和EP-2/H1478)PCR可准确地鉴定出马鹿和梅花鹿等。结论特异引物PCR适宜马鹿、梅花鹿等珍贵中药材的鉴定。     

柴胡及其常见伪品的DNA指纹鉴定

目的:建立柴胡及其伪品DNA指纹鉴别方法.方法:采用改良的CTAB法提取柴胡及常见伪品的干根基因组DNA,设计鉴别正品柴胡及其伪品的特异性引物,利用PCR扩增技术对正品柴胡及其伪品进行DNA指纹鉴定.结果:改良的CTAB法提取出大小为23 kb的基因组DNA条带,纯度在(1.71±0.11),且经特异引物PCR扩增,只有正品柴胡能扩增出173bp大小的片段,而伪品未能扩增出目的片段.结论:柴胡DNA具有特异性指纹特征,可作为市售柴胡的真伪鉴定依据.     

中药材鹿鞭的分子鉴定研究

目的 :建立中药材鹿鞭的分子分类学鉴定试剂盒。方法 :根据对不同产地梅花鹿、马鹿、白唇鹿、水鹿线粒体DNA进行PCR扩增和序列测定 ,并与常见伪充药材来源动物线粒体DNA同位置序列比较 ,找到该 4个鹿种的特征片段 ,经过实际检验 ,筛选出合适片段作为鹿的种属特异性PCR引物。结果 :该引物与相关试剂组成试剂盒后 ,可用于中药材鹿鞭与常见伪充药材牛鞭、驴鞭等的鉴别。结论 :用分子分类学方法鉴别中药材鹿鞭 ,具有科学、稳定、准确、简便等特点 ,值得推广。     

桃仁及苦杏仁DNA检测试剂盒的研制与评价

目的 本研究旨在研制一种快速检测试剂盒,鉴定桃仁及苦杏仁DNA成分,并对其进行性能评价。方法 建立DNA提取方法,确保有效提取桃仁及苦杏仁基因组DNA,紫外分光光度法测其浓度及纯度。美国国立生物技术信息中心(NCBI)查询并比对桃仁及苦杏仁的ITS序列,针对特异性位点,NCBI Primer-Blast设计种属特异性引物。建立并优化聚合酶链式反应(PCR)检测体系,DH5α感受态细胞克隆特异性片段,测序验证体系准确性,制备阳性对照。组装DNA检测试剂盒,对其特异性、稳定性及重复性进行评价,并对市售桃仁和苦杏仁样品进行检测。结果 所建立DNA提取方法,提取效率高,DNA质量浓度高于100 ng·μL^-1,A_260/280处于1.80～2.10之间。该试剂盒可准确鉴定桃仁及苦杏仁DNA成分,检测灵敏度高,特异性强,最低检测限为1 ng·μL^-1。反复冻融20次后仍可有效检测,可于-20 ℃保存1年。20个市售样品中检出1个桃仁伪品及2个苦杏仁伪品,其余均为正品。结论 桃仁及苦杏仁DNA检测试剂盒特异性强、灵敏性高、稳定性好,具有良好重复性,可快速鉴定桃仁及苦杏仁,为桃仁中药材市场质量安全管理提供有力支持。     

基于COI 条形码的鹿类中药材DNA 条形码分子鉴定

目的：利用COI 序列建立统一的鹿类药材分子鉴定方法。方法：对鹿茸、鹿角、鹿鞭、鹿筋、鹿尾、鹿胎分别进行DNA 提取、COI 序列扩增和序列测定,构建鹿类药材COI 序列数据库,并对市售鹿类药材进行调查分析。结果：所有鹿类药材均可以使用COI 通用引物进行PCR 扩增和测序;鹿类药材COI 序列数据库包含8 个物种101 份样品,物种之间相互区分明显;市售40 份药材中18 种为药典规定物种,22种为非药典规定物种。结论：COI 序列的DNA 条形码分子鉴定方法可以作为鹿类药材的统一鉴定方法,并为市售鹿类药材的鉴定提供科学依据。     

中药穿山甲的DNA分子鉴定研究

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。     

貂心线粒体mtDNA鉴定及特征分析

 目的探讨貂心肌线粒体DNA(mtDNA)的特征,建立中药利心丸中貂心的分子遗传学鉴定方法。方法从新鲜的貂心组织提取mtDNA,采用DNA限制性内切酶片段长度多态性(mtDNA-RFLP)分析技术和聚合酶链反应(PCR)扩增细胞色素b基因部分序列片段。结果新鲜的貂心组织可提取完整的mtDNA,并能扩增出310 bp大小的Cytb基因,与鸡心脏mtDNA限制性内切酶酶切图谱有差异。结论2种方法可以作为利心丸中貂心物种的区分提供方便有效的分子标记。     

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??OBJECTIVE To develop a DNA detection kit for identification of Fetus Cervi, evaluate the quality indexes including specificity, stability, sensitivity and repeatability, and inspect the qualities of commercial Fetus Cervi samples. METHODS The Fetus Cervi DNA test kit was developed and modified using the classical DNA extraction and PCR identification method. The genomic DNAs of Fetus Cervi and counterfeit goods were extracted by the kit and PCR technique was performed to identify the authenticity. The purity of DNA was detected by UV spectrophotometer. Finally, commercially available Fetus Cervi samples were verified. RESULTS The kit was proved to be effective after 20 times of repeated frozen-thawing and it could be stored at -20 ?? for 1 year. The DNA could be detected when the primary solution was diluted by 200 times. The specificity test confirmed that the 15 samples were authentic Fetus Cervi, and 7 were counterfeit. The specificity of the kit was 100%. The repetitive experiments showed that the average intra-assay CV% and inter-assay CV% of the kit were 2.32% and 2.56%, respectively. CONCLUSION The amount and purity of the nucleic acids extracted by the DNA detection kit can meet the requirement for identification of Fetus Cervi, and the kit has good specificity, high sensitivity and good stability, so it is suitable for the rapid detection of Fetus Cervi.     

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??OBJECTIVE To develop Zaocys dhumnades DNA extraction and detection kit, evaluate the specificity, stability and repeatability of the kit, and investigate the quality of commercial black tip snake products using the kit. METHODS Using the modern DNA fingerprint technology, the pharmacopoeia Zaocys dhumnades DNA detection method was optimized and improved, and Zaocys dhumnades DNA extraction and detection reagent was developed to obtain the tip snake PCR detection kit. The performance parameters of the kit were evaluated. The kit was used on 18 commercial black tip snake products randomly sampled from Beijing, Tianjin, Changchun and Jilin City.The results were compared with those got by using pharmacopoeia method of DNA test. RESULTS Zaocys dhumnades DNA testing kit was still valid after repeated freezing and thawing for 5, 10 and 20 times. Among the 18 commercial black tip snake samples 14 were quality goods, and four were adulterants. Repeated experiments of specificity, stability and repeatability displayed exactly the same results . The test result of the kit method was consistent with the pharmacopoeia method. And the kit method could fulfil the DNA testing task in ordinary laboratories. CONCLUSION Zaocys dhumnades DNA testing kit can identify tip snake products conveniently, accurately and effectively, which can be applied in the rapid detection of Zaocys dhumnades DNA.     

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??OBJECTIVE To develop a rapid DNA detection kit for DNA extraction and PCR identification of Panax ginseng C.A. Mey. METHODS The classical DNA extraction and PCR identification METHODS for Panax ginseng C.A. Mey were modified, and the compositions and reaction conditions of the kit were determined. In addition, the specificity, stability, sensitivity, and repeatability of the kit were evaluated. The genomic DNAs of genuine and counterfeit ginseng goods were extracted by the kit and PCR was performed to identify the authenticity. The purity of the extracted DNA was detected by UV spectrophotometry. Finally, commercially available ginseng samples were verified. RESULTS The purity of the genomic DNA extracted by the kit was (1.73??0.13)(OD₂₆₀/ OD₂₈₀), and a fragment between 150 and 200 bp could be amplified only from authentic Panax ginseng C.A. Mey. The specificity of the kit was 100%. The repetitive experiments showed that the average intra-assay CV% and inter-assay CV% of the kit were 2.38% and 2.62%, respectively. The DNA in solutions diluted by 200 times could still be detected. Stability experiment proved that repeated freeze-thawing for 20 times had no significant effect on the activity of this kit and the test sample could be stored at -20 ?? for one year. The specificity test confirmed that 8 samples among the 10 commercial products were genuine, and 2 were counterfeit. CONCLUSION The nucleic acid extraction and purity of the DNA detection kit can meet the requirement for identification of Panax ginseng C.A. Mey.The kit has good specificity, high sensitivity, and good stability, so it is suitable for the rapid detection of Panax ginseng C.A. Mey.     

线粒体COI和CYTB基因在虫草属物种寄主昆虫鉴定中的适用性分析

目的：利用DNA条形码技术对冬虫夏草、凉山虫草、亚香棒虫草、戴氏虫草等物种的寄主昆虫分别通过COI序列和Cytb序列进行条形码分析,探索虫草属物种寄主昆虫鉴定序列的适用性。方法：通过DNA提取、PCR、电泳检测和序列测定,分别获得26份虫草寄主虫体的COI序列和Cytb序列,再用拼接和分析软件进行序列的比对和聚类分析。结果：COI序列和Cytb序列都能很好地区分鉴定虫草类各物种,但COI序列的扩增成功率大于Cytb基因,同时在COI基因片段中共检测到129个多态位点,变异率为19.51%;在Cytb基因片段中共检测到113个多态位点,变异率为26.10%。COI序列的简约信息点多于Cytb序列,且基因变异率低于后者。结论：本研究建议COI序列更适用于虫草寄主昆虫的DNA条形码研究。     

中药材鳖甲的位点特异性PCR鉴定研究

目的 本研究旨在建立一种简便、实用的鳖甲DNA分子鉴定方法。方法 根据中华鳖和鳖甲混淆品原动物的125rRNA基因片段的序列数据库,找出中华鳖与其它鳖科动物有明显区别的位点,设计1对能特异性鉴别中华鳖的引物,然后利用鳖甲中残存的DNA,通过PCR拉增,即可准确鉴别鳖甲。结果 用这对引物对不同来源的10块鳖甲进行鉴定结果表明,其中有3块为伪品,结果与DNA序列分析鉴定一致。还测定了斑龟和鳖甲一伪品12SrRNA基因片段序列。结论 该引物配置药材鉴定试盒可在鳖甲类药材鉴定使用。