基于巢式PCR方法联合Sanger测序技术对大黄庶虫丸中水蛭成分真伪的鉴定

目的 基于DNA条形码技术建立一种可以实现对大黄庶虫丸中水蛭成分真伪品鉴定的方法。方法 首先以COⅠ通用引物对大黄庶虫丸中成药DNA进行一轮聚合酶链式反应(PCR)扩增,随后采用自行设计和筛选出的水蛭通用引物对一轮PCR反应液进行二轮PCR扩增,联合Sanger测序技术,对二轮PCR扩增产物测序,最后对测序结果峰图采用seqman软件进行序列拼接,将拼接后的DNA序列在NCBI数据库中用BLAST在线比对软件进行比对判定,进而鉴定水蛭成分真伪。结果 本实验建立的巢氏PCR方法联合Sanger测序技术通过了对9份经COⅠ通用引物鉴定的水蛭干药材的验证,结果验证两方法鉴定结果一致。同时采用本研究建立的巢氏PCR方法联合Sanger测序技术对收集的14批大黄庶虫丸进行鉴定,共有5批鉴定出含有伪品水蛭成分。结论 本实验建立的的巢氏PCR方法联合Sanger测序技术能够实现对大黄庶虫丸中水蛭成分真伪品的鉴定,对中成药的质量控制具有一定的意义。     

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。     

二株具有抗菌活性红树林土壤放线菌的分离与鉴定

目的 了解广西北海红树林土壤放线菌的抗菌活性.方法 采用海水配制高氏培养基分离广西北海红树林土壤中的放线菌.提取二株典型放线菌发酵液进行抗菌实验,同时提取其总DNA,用放线菌通用引物对16S rDNA进行PCR扩增,对获得的扩增结果进行DNA序列测定.结果 提取到的二株典型放线菌发酵液具有抗菌活性,同时将二株典型放线菌菌株的16S rDNA,经测序后对测序结果进行比对显示,所获的BH200951和BH200954菌株与链霉菌属形态学特征、生理生化特征和基于16S rDNA (16S rRNA)基因序列的系统发育分析,BH200951被鉴定为链霉菌属的有效发表种链霉菌的菌株Streptomyces aurantiogriseus AY999773的变种,BH200954初步被鉴定为链霉菌属的有效发表种仙台链霉菌.结论 分离来自广西北海红树林土壤中的二菌株其发酵产物有很强的抗菌活性,具有进行新药开发利用的潜力.     

基于cp DNA序列和ISSR分子标记的甘肃祁连山地区麻花秦艽变异与遗传分化分析

目的 采用叶绿体基因(cp DNA)序列和ISSR分子标记技术对甘肃祁连山地区麻花秦艽进行序列变异和遗传分化分析。方法 利用试剂盒提取法提取麻花秦艽的基因组DNA,利用筛选出的trnS-trnG和rpl20-rps12引物进行扩增并测序,通过软件Chromas、ContigExpress对得到的序列进行校正、拼接,利用MegaX、DanSP5、GenALEx软件对拼接后的序列进行序列特征分析,最后利用PopART软件得到麻花秦艽的单倍型网状进化图。结果 18个麻花秦艽居群共计114个个体被成功扩增和测序;2个cp DNA片段经拼接后的长度为1 246 bp,共有676个多态性变异位点,459个单一突变位点、217个简约信息位点和107个插入-缺失位点;单倍型41个,其中特有单倍型34个;Tajima′s D检验在P>0.05水平上不显著,整体遵循中性进化模型。居群内变异百分比为89%,说明遗传变异主要存在于居群内;Fst=0.114(P<0.05),Nm=3.889说明居群间分化程度较低,居群间具有较强的基因交流。遗传距离与地理距离相关性分析r=-0.094(P<0.05),说明遗传距离与地理距离不具有明显的相关性。结论 甘肃祁连山地区麻花秦艽在物种水平上遗传多样性较高,具有丰富的单倍型类型,居群的遗传变异主要来自于遗传漂变。     

中华地鳖虫肠道内生真菌的分离鉴定及抑菌活性研究

目的:研究中华地鳖虫肠道内生真菌及其抑菌活性,为发现新型活性天然产物提供一条重要的途径。方法:采用平板分离法从地鳖虫肠道内分离内生真菌,利用滤纸片法对其进行抑菌活性研究,并对菌株的总DNA进行提取,利用通用引物ITS1和ITS4对菌株18S rDNA ITS序列进行扩增和测序,将测序结果进行同源性比对分析,确定活性菌株的分类地位。结果:从中华地鳖虫肠道中共分离出8株内生真菌。其中,菌株DB-1对金黄色葡萄球菌、枯草芽孢杆菌和白色念珠菌都有一定的抑制作用,经比对分析后鉴定菌株DB-1为深绿木霉Trichoderma atroviride。结论:中华地鳖虫肠道内具有抑菌活性的内生真菌资源可做进一步研究利用。     

一株耐甲氧西林金黄色葡萄球菌MS4的全基因组测序结果分析

目的:分析创口分离一株耐甲氧西林金黄色葡萄球菌(MRSA)的全基因组序列特点。方法:将深圳大学南山区人民医院一例创口脓液分离菌株采用BD Phoenix TM100自动细菌鉴定/药敏系统鉴定;用E-Test试验测定其利奈唑胺(LZD)最低抑菌浓度(MIC)值;扩增细菌提取细菌总DNA,应用多重PCR扩增及DNA测序方法行SCCmec-spaMLST分型;并采用Illumina Miseq结合第三代测序技术分别构建了Illumina Miseq PE文库(~500 bp)和Pac Bio文库(8~10 kb),普通PCR覆盖gap,对获得的测序数据进行质控后利用生物信息学分析手段完成该菌株的全基因组完成图绘制。结果:经鉴定此菌株为耐甲氧西林金黄色葡萄球菌(命名为MS4)。药敏结果显示该菌株对头孢西丁、氨苄西林、甲氧西林、青霉素P、阿莫西林/克拉维酸耐药,对利奈唑胺等其余抗菌素敏感。E-Test提示LZD MIC值为0.094 ug/m L。SCCmecspa-MLST分型结果显示该菌株属于III-t 3592-ST59型。MS4基因组包含2709797 bp,GC含量33.5%;通过注释后总共有2475个基因,11个t RNA编码基因,3个完整的r RNA基因编码操纵子,并包括mec A等耐药基因和γ-溶血素、溶血素III、烯醇酶、肠毒素等多种毒力因子,申请获得Genebank号CP009828。结论:完成了一株创口脓液来源MRSA的基因分型和全基因组完成图绘制,为MRSA基因组比对和系统进化发育分析提供了模板序列。     

亚硝胺类基因毒性杂质专项应急检验管理的一些思考

目的 进一步提高药品应急检验应对能力。方法 采用回顾性研究的方法,对江苏省食品药品监督检验研究院2019年亚硝胺类基因毒性杂质专项应急检验全过程工作进行梳理。结果 总结了专项应急检验中各环节的处置流程、管理特点和难点,提炼处置经验教训。结论 应急检验工作是对检验机构应急响应能力、技术实力的全面考验。需从事前、事中和事后制订系统的应对策略,构建完善的药品应急检验管理体系。     

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。     

应用PCR-RFLP方法鉴定梅花鹿茸与马鹿茸

目的 建立一种应用限制性片段长度多态性技术鉴别梅花鹿茸与马鹿茸的方法。方法 提取各种鹿茸样品的基因组DNA,通过聚合酶链式反应(polymerase chain reaction,PCR)对正品鹿茸的特异性片段进行扩增,鉴别鹿茸的真伪,然后对正品鹿茸利用动物通用引物进行PCR扩增,并通过限制性片段长度多态性分析进一步对正品鹿茸的种属来源进行确证。结果 通过对特异性片段的PCR扩增,可将梅花鹿茸及马鹿茸与驯鹿、麋鹿、新西兰鹿等伪品鹿茸加以区分,通过对限制性内切酶Msp I 进行酶切后的片段长度进行分析,可进一步区分梅花鹿茸与马鹿茸。结论 本实验建立了一种基于限制性片段长度多态性技术快速、准确的鉴别鹿茸真伪及种属鉴定的方法,为解决中药易混品种难以鉴定的问题提供了又一个新方法。     

108份血液细菌培养结果及耐药性分析

目的:了解败血症患者病原菌分布及常见病原菌耐药情况。方法:采用法国梅里埃VITEK SYSTMS—ATB半自动细菌仪对近年来血培养108株病原菌进行鉴定和药敏试验。结果:分离数占前5位的菌株依次为表皮葡萄球菌、溶血葡萄球菌、大肠埃希菌、金黄色葡萄球菌、甲型副伤寒。结论:菌血症仍以凝固酶阴性葡萄球菌为主,肠杆菌科比例较高,临床需及时掌握血培养的细菌分布及药敏结果,以便于合理使用抗生素。     

基于PCR-核酸试纸条快速鉴定鹿茸及其伪品的实验研究

目的 通过聚合酶链式反应(polymerase chain reaction, PCR)与核酸试纸条相结合的方法,实现鹿茸真伪的可视化检测。方法 采用十二烷基硫酸钠(sodium dodecylsulfate, SDS)碱变性法提取正品及伪品鹿茸样品基因组DNA,通过查找细胞色素C氧化酶亚基I(COI)特异性位点,应用Primer 3.0软件设计鹿茸特异引物,摸索PCR最佳反应体系及条件,建立PCR-核酸试纸条及琼脂糖凝胶电泳鉴定鹿茸真伪的最优条件。同时,通过克隆测序,验证鹿茸真伪鉴定的准确性。结果 本实验中正品鹿茸在PCR-核酸试纸条上均出现两条带,阴性对照及伪品出现一条带,与琼脂糖凝胶电泳结果吻合,且试纸条检测的灵敏度可达1 ng·μL^-1。对9个市售鹿茸样品检测,正品鹿茸5个,合格率为45%。结论 自主构建的PCR-核酸试纸条检测方法简单、快速、特异性较好,可在较短时间内实现鹿茸真伪的可视化检测,检测结果稳定,为中药材鉴定提供又一新的方法。     

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??OBJECTIVE To establish the high-performance thin layer chromatographic(HPTLC)fingerprints of volatile oil and flavonoids in Alpinia katsumadai Hayata so as to provide scientific information for its quality control, and determine the fingerprints similarity of with its related species. METHODS The separation was performed on the pre-coated HPTLC GF₂₅₄ silica gel plates. The volatile oil was developed with solvent system of toluene-ethyl acetate(9??1). The relative humidity was 18%. The spots were visualized with 5% vanillin sulfuric acid solution. The flavonoides compounds were developed with solvent system of toluene-ethyl acetate-acetic acid (8??1.5??0.5). The spots were visualized with 5% aluminium chloride ethanol solution which needed to be observed at 365 nm. The common patterns of HPTLC fingerprints were obtained by CHROMAP 1.5 solution software, and authentication and quality assessment were performed by similarity and principle component analysis. RESULTS The common patterns of the volatile oil and flavonoids consisted of 10 characteristic peaks and 7 characteristic peaks, respectively. Eucalyptol and alpinetin were identified by chemical reference substances. CONCLUSION The qualities of Alpinia katsumadai Hayata collected from different areas are not distinctly different. Obvious difference exists in the chemical compositions of the volatile oil and flavonoids between Alpinia katsumadai Hayata and its counterfeit and other related species. This method is simple and rapid, which can serve as an effective identification and quality assessment method for Alpinia katsumadai Hayata.     

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??OBJECTIVE To establish the fingerprint identification and assay for the quality analysis of processed Aconitum sinomontanum Nakai from different areas. METHODS HPLC Gradient elution method was developed to establish the fingerprints for processed Aconitum sinomontanum Nakai, and the fingerprints were analyzed and compared by Chinese Materia Medica (CMM) Fingerprint Similarity Evaluation System (2012 edition), principal component analysis (PCA) and cluster analysis (CA). RESULTS The common fingerprint for processed Aconitum sinomontanum Nakai fingerprints was established, and 18 common fingerprint peaks were identified. The similarity was greater than 0.90 among 10 batches of processed Aconitum sinomontanum Nakai medicinal herbs, and the contents of lappacontine and ranaconitine were determined. The samples from different areas could be classified into four groups, which reflected the quality characteristics of 10 batches of processed Aconitum sinomontanum Nakai from different areas. Four main components with cumulative contribution rate of 88.824% were selected by PCA, and seven chemical components were identified as the ones to determine the quality of processed Aconitum sinomontanum Nakai. CONCLUSION This method, with good reproducibility and strong characteristics, can be used for the comprehensive quality evaluation of processed Aconitum sinomontanum Nakai.     

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??OBJECTIVE To identify Polygonum chinensis and its adulterants by ITS2 sequences. METHODS Total genomic DNA of P. chinensis was extracted using the plant genomic DNA kit. The internal transcribed spacer 2(ITS2) regions were amplified. The variable site of ITS2 regions were analysed through MEGA 6.0 software. The intra-versus inter-specific genetic distances of the ITS2 regions was calculated based on the kimura 2-parameter(K2P) model. Neighbor-Joining phylogenetic trees were constructed using MEGA6.0. RESULTS The intraspecific variation of P. chinensis was small. However, the interspecific variation of P. chinensis and its adulterants was small distinct. The secondary structure of ITS2 of P. chinensis and its adulterants has significant difference. NJ trees can identify P. chinensis and its adulterants. CONCLUSION ITS2 Regions can be used to authenticate P. chinensis and its adulterants which provide new METHODS for the identification of P. chinensis. The standard DNA barcodes of P. chinensis are establishment which would lay the foundation of identification and safety clinical drug application of P. chinensis.     

新型眼用硝酸毛果芸香碱六角液晶凝胶的制备和评价

目的 本研究以硝酸毛果芸香碱(PN)为模型药,以植烷三醇(PHYT)/三辛酸甘油酯(TAG)/水三元系统制备六角相液晶(HⅡ)凝胶,通过眼部给药提高PN的眼用生物利用度.方法 采用偏光显微镜(CPLM)、小角X射线散射仪(SAXS)和流变学对HⅡ凝胶的内部结构进行表征;通过体外释放和离体角膜渗透对HⅡ凝胶的体外行为进行...     

环介导等温扩增技术在西洋参鉴定中的应用

目的 探讨利用环介导等温扩增技术(loop-mediated isothermal amplification,LAMP)技术鉴别西洋参的方法.方法 针对西洋参的CYP450基因设计特异性引物,应用RT-LAMP(real time-LAMP)、琼脂糖凝胶电泳和可视化检测3种方法对LAMP检测方法进行优化,并对其灵敏性...     

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??OBJECTIVE To study the HPLC fingerprint of Swertia patens, the most commonly used substitute of Swertiae Mileensis Herba recorded in Chinese Pharmacopoeia, compare the major chemical constituents between the two kinds of herbs, and provide scientific evidence for their identification and quality control. METHODS HPLC fingerprint method was used to analyze 12 batches of S. patens and 5 batches of Swertiae Mileensis Herba. Similarity evaluation method and clustering analysis method were introduced to compare the HPLC chromatograms of them. RESULTS The repetition,stability, and precision of the fingerprint method were good. A total of 13 common peaks were confirmed. The similarities of the chromatograms of 17 batches of Swertia were greater than 0.94. CONCLUSION Different batches of S. patens and Swertiae Mileensis Herba have high similarity. It is possible for S. patens to take place of Swertiae Mileensis Herba.     

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??OBJECTIVE To prepare and characterize sinomenine liquid crystal gel, study the rheological properties and in vitro transdermal properties and compare with sinomenine ointment and sinomenine hydrophilic gel. METHODS Sinomenine liquid crystal gel was prepared by using phytanetriol-water system and then characterized by polarized light microscope(PLM)and small angle X-ray diffraction(SAXS). Using DHR-2 rheometer,the rheological properties of sinomenine liquid crystal gel were investigated and compared with those of sinomenine ointment and sinomenine hydrophilic gel. Modified Franz diffusion cell was used for in vitro transdermal experiment and the in vitro transdermal properties were compared with sinomenine hydrogels and ointments. RESULTS The appearance of sinomenine liquid crystal gel was colorless, clear and transparent, and showed dark field under PLM. SAXS showed cubic phase. The rheological parameters were good. The steady-state infiltration rates of sinomenine liquid crystal gel, ointment and hydrophilic gel were 153.93, 119.99, and 106.89 ??g??cm^-2??h^-1,and their 48 h cumulative permeation amounts(%)were 93.76%,91.55%, and 87.60%, respectively. CONCLUSION PLM and SAXS can be used to characterize the liquid crystal gel.The prepared sinomenine liquid crystal gel has good appearance and suitable rheology, and its infiltration rate and 48 h cumulative permeation amount are superior to those of ointment and hydrophilic gel, which provides theoretical reference for sinomenine percutaneous administration.