应用16SrRNA基因序列分析快速鉴定临床标本中的革兰氏阳性杆菌

目的探索一种快速鉴定临床标本中革兰氏阳性杆菌的方法。方法利用PCR技术扩增待检菌株的16SrRNA基因序列,通过分析待检菌株的16SrRNA基因序列对其进行鉴定。结果5株待检菌株的16SrRNA基因序列均成功扩增,其中4株的16SrRNA基因序列与基因库中已注册的核酸序列相似率达99．9％以上,将其鉴定到种的水平,1株的16SrRNA基因序列与基因库中雷弗森菌属的核酸序列相似率为97．09％,将其鉴定为雷弗森菌属。结论应用16SrRNA基因序列分析可快速、准确地鉴定临床标本中的革兰氏阳性杆菌。     

鲍曼不动杆菌老年患者分离株中发现16S rRNA甲基化酶基因新亚型

目的了解鲍曼不动杆菌老年患者分离株16SrRNA甲基化酶和氨基糖苷类修饰酶基因型。方法采用PCR检测20株鲍曼不动杆菌老年患者分离株12种16SrRNA甲基化酶和氨基糖苷类修饰酶基因。结果7株检出16SrRNA甲基化酶基因（armA），19株检出氨基糖苷类修饰酶基因[其中aac（3）-I阳性率为95％，ant（3″）-I为95％，aac（3）-II为40％，aac（δ′）-Ib为15％）]。6号株armA基因测得序列翻译成氨基酸序列与美国核酸库（GenBank）已登录的armA氨基酸不同，为新亚型。结论本组鲍曼不动杆菌老年患者分离株95％携带16SrRNA甲基化酶和氨基糖苷类修饰酶基因。     

PCR扩增rRNA基因在细菌菌种鉴定中的应用

PCR技术以其快速及准确的优点已广泛应用于微生物检测与菌种鉴定中。本文重点从16SrRNA序列、16S—23S rRNA间隔序列、5S rRNA序列及tRNA基因序列扩增四个方面介绍了近年来PCR技术在细菌菌种鉴定中的应用。     

5SrRNA基因间隔区DNA序列在穿心莲道地性中的可行性研究

目的探讨5SrRNA基因间隔区DNA序列在穿心莲道地性研究中的可行性。方法提取不同产地穿心莲总DNA后,PCR特异扩增5SrRNA基因间隔区,采用荧光标记末端终止子双脱氧末端终止法测序,对测序结果进行多序列联配分析。结果成功得到321bp的5SrRNA基因间隔区碱基序列,经过多序列联配分析发现,不同产地穿心莲的此段5SrRNA基因间隔区DNA序列之间没有差异。结论5SrRNA基因间隔区DNA序列不适合用于研究穿心莲药材的道地性。     

儿童近端小肠粘膜相关菌群分布研究

目的研究儿童近端小肠粘膜相关菌群种类和分布。方法取儿童近端小肠粘膜活检组织,提取总DNA,经PCR扩增后对16SrRNA基因扩增片段克隆,并对克隆片段进行DNA序列测定,根据序列结果分析细菌组成。同时在不同培养条件下进行细菌培养及鉴定。结果16SrRNA基因测序结果表明儿童近端小肠粘膜相关菌群包括6个细菌门类,厚壁菌门、变形菌门和放线菌门占总细菌的88.8%;拟杆菌门、梭杆菌门和TM7占11.2%。链球菌属和奈瑟菌属为儿童近端小肠粘膜相关菌群的主要菌属;韦荣菌属、孪生菌属、放线菌属、罗氏球菌属、嗜血菌属、普雷沃菌属和颗粒链菌属占较高比例。细菌培养结果与16SrRNA基因测序结果类似。结论儿童近端小肠粘膜具有独特的菌群定殖分布。     

应用PCR方法检测重症合并感染病人血中细菌16SrRNA基因的临床价值

目的探讨病人血中细菌16SrRNA基因对临床诊断重症合并感染的意义。方法40例APACHEⅡ评分≥12分重症合并感染的病人，抽取其外周血，应用PCR技术检测细菌16SrRNA基因同时做血细菌培养。对PCR和血培养检测的结果进行比较。30例正常对照组检测其血中细菌16SrRNA基因。结果40例重症合并感染的病人中有13人用PCR法检出细菌16SrRNA的基因，阳性率为32．5％。血细菌培养阳性的6人，阳性率仅为15％。血细菌培养阳性者其PCR结果全部为阳性。结论用PCR方法检测重症合并感染病人血中细菌16SrRNA基因具有灵敏、快速、不受使用抗生素的影响，与临床诊断吻合度高等优点，弥补与丰富了微生物学的检测方法，为准确认识和救治重症合并感染提供科学依据。     

基于COⅠ与16S rRNA基因对广地龙的DNA分子鉴定研究

目的:建立一种快速、准确和标准化的广地龙DNA分子标记鉴别方法。方法:测定了5个不同居群广地龙的线粒体细胞色素酶亚单位(CO)Ⅰ和16S rRNA基因序列,采用CodonCode Aligner进行序列拼接,通过下载GenBank地龙原动物的COⅠ与16S rRNA序列,采用MEGA4.1计算广地龙及其伪品地龙的种内、种间的K2P遗传距离,并基于K2P模型构建NJ和MP树。结果:COⅠ变异位点、信息位点均高于16SrRNA,COⅠ基因无插入和缺失,16S rRNA存在4个插入和缺失。COⅠ和16S rRNA序列种间遗传距离均明显大于种内,COⅠ和16S rRNA基因均能将广地龙从其他地龙或蚯蚓物种鉴别开来。结论:获得的广地龙COⅠ和16S rRNA序列可为动物性中药材地龙的分子水平鉴定提供参考,为动物性中药材DNA条形码数据库积累了相关信息数据。     

香港特有止泻中药材红党参的化学和分子生物学鉴定

目的鉴定香港特有止泻中药材红党参的基源植物和表面红色物质。方法将红党参的5SrRNA基因间隔区序列和HPLC色谱指纹谱与正品党参5SrRNA基因间隔区序列和HPLC色谱指纹谱进行比较研究;将红党参表面红色物质的X-衍射光谱与中药赤石脂X-衍射光谱进行比较研究。结果红党参的HPLC色谱指纹谱和党参药材具有相同的特征色谱峰;红党参的5SrRNA基因间隔区序列与党参正品之一的素花党参（Codonopsis pilosula var.modesta）的5SrRNA基因间隔区序列最为相似;红色物质的X-衍射光谱与中药赤石脂X-衍射光谱完全相同。结论红党参的基源是素花党参（Codonopsis pilosula var.modesta）的根,红党参表面的红色物质是传统中药赤石脂（Halloysitum Rubrum）。红党参是素花党参根用中药赤石脂炮制而成,科学名可定为Radix Codonopsis Praeparata Halloysita Rubra。     

中药材龟甲的分子鉴定研究

用PCR产物直接测序法对中药材龟甲(板)进行鉴别。从乌龟 Chinemys revesii 和其他20种产地为中国或东南亚国家的龟类的组织材料中提取DNA,扩增约110bp的线粒体12SrRNA,基因片段并进行序列分析,构建了21种龟类的12SrRNA基因片段序列数据库。序列比较的结果表明乌龟与其它20种龟类的这段序列均有差别,序列差异在3.7～15.7%之间。从江苏省药品检验所提供的19块龟甲检品上各取样0.1～0.5g提取 DNA,扩增与上述相同的基因片段,与构建的数据库进行比较,结果表明19块龟甲中只有3块的原动物为乌龟,其余的龟甲均为混淆品。本文的结果为药材龟甲的鉴定找到了有效、可靠的分子遗传标记方法。     

海绵细菌B25W的鉴定及其活性物质的初步研究

分离自繁茂膜海绵(Hymeniacidonperleve)的细菌B25W经16SrRNA序列分析,鉴定为解淀粉类芽孢杆菌(Paenibacillusamyloliquefaciens)。解淀粉类芽孢杆菌产生的生物活性物质对植物和人类的致病真菌葡萄孢菌(Botrytisfuckeliana)、镰刀菌(Fusariumsolani)、紫青霉(Penicillumpurpurogenum)、稻瘟霉(Piriculariaoryzae)、白念珠菌(Candidaalbicans)有很好的拮抗作用,是一种对热和酸稳定的水溶性物质。     

Temporal Patterns of Bacterial and Viral Communities during Algae Blooms of a Reservoir in Macau

Compositions of microbial communities associated with blooms of algae in a storage reservoir in Macau, China were investigated between 2013 and 2016. Algae were enumerated by visible light microscopy. Profiles of organisms in water were examined by 16S rRNA sequences and viral metagenomics, based on next generation sequencing. Results of 16S rRNA sequencing indicated that majority of the identified organisms were bacteria closely related to Proteobacteria, Cyanobacteria, Verrucomicrobia, Bacteroidetes, and Actinobacteria. Metagenomics sequences demonstrated that the dominant virus was Phycodnavirus, accounting for 70% of the total population. Patterns of relative numbers of bacteria in the microbial community and their temporal changes were determined through alpha diversity indices, principal coordinates analysis (PCoA), relative abundance, and visualized by Venn diagrams. Ways in which the bacterial and viral communities are influenced by various water-related variables were elucidated based on redundancy analysis (RDA). Relationships of the relative numbers of bacteria with trophic status in a reservoir used for drinking water in Macau, provided insight into associations of Phycodnavirus and Proteobacteria with changes in blooms of algae.     

一株尿液标本分离获得Actinotignum schaalii的鉴定及生物学特性分析#br#

目的　探讨Actinotignum schaalii的鉴定方法及生物学特性，为临床诊断Actinotignum schaalii引起的泌尿系感染提供依据。方法　收集1例于2020年1月在天津中医药大学第一附属医院住院泌尿系感染患者的尿液标本，对其进行细菌培养及革兰染色，观察细菌形态特点。用基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）及16S rRNA基因测序技术进行鉴定。所得序列与GenBank数据库中的序列进行比对，并进行系统发育分析。结果　该病例由Actinotignum schaalii引起泌尿系感染，为国内首例报道。经MALDI-TOF MS与16S rRNA基因检测鉴定为Actinotignum schaalii，属于放线棒菌属，为兼性厌氧、革兰阳性杆菌。判断其为引起泌尿系感染的新型致病菌。结论　Actinotignum schaalii可能是引起泌尿系感染的新型致病菌。     

关节液中细菌16s rRNA与23s rRNA诊断膝关节置换术后感染的价值

目的 探讨利用关节液中细菌16s rRNA与23s rRNA诊断全膝关节置换术后感染的效率及两种基因诊断方法的差异.方法 对33例无菌性松动及19例假体周围感染行人工膝关节翻修的患者,通过RT-PCR检测关节液中细菌16s rRNA、23s rRNA保守基因片段诊断假体周围感染.比较两种诊断策略的敏感性、特异性、阳性预测值、阴性预测值及准确性.结果 以国外关于假体周围感染诊断方法的文献判定假体周围感染,利用16s rRNA进行诊断的敏感性78.8％,特异性93.9％,阳性预测值88.2％,阴性预测值为88.6％,准确性为88.5％;而采用23s rRNA扩增方法诊断的敏感性、特异性、阳性预测值、阴性预测值及准确性分别为68.4％、78.8％、65.0％、81.2％和75.0％.两种基因诊断的各指标比较差异均无统计学意义(P>0.05).结论 通过检测关节液中细菌16s rRNA或23s rRNA诊断人工膝关节置换术后感染,具有较高的诊断效率,且两者差异无统计学意义.     

Mangrove trees affect the community structure and distribution of anammox bacteria at an anthropogenic-polluted mangrove in the Pearl River Delta reflected by 16S rRNA and hydrazine oxidoreductase (HZO) encoding gene analyses

Anaerobic ammonium oxidizing (anammox) bacterial community structures were investigated in surface (1–2 cm) and lower (20–21 cm) layers of mangrove sediments at sites located immediately to the mangrove trees (S0), 10 m (S1) and 1000 m (S2) away from mangrove trees in a polluted area of the Pearl River Delta. At S0, both 16S rRNA and hydrazine oxidoreductase (HZO) encoding genes of anammox bacteria showed high diversity in lower layer sediments, but they were not detectable in lower layer sediments in mangrove forest. S1 and S2 shared similar anammox bacteria communities in both surface and lower layers, which were quite different from that of S0. At all three locations, higher richness of anammox bacteria was detected in the surface layer than the lower layer; 16S rRNA genes revealed anammox bacteria were composed by four phylogenetic clusters affiliated with the “Scalindua” genus, and one group related to the potential anammox bacteria; while the hzo genes showed that in addition to sequences related to the “Scalindua”, sequences affiliated with genera of “Kuenenia”, “Brocadia”, and “Jettenia” were also detected in mangrove sediments. Furthermore, hzo gene abundances decreased from 36.5 × 10⁴ to 11.0 × 10⁴ copies/gram dry sediment in lower layer sediments while increased from below detection limit to 31.5 × 10⁴ copies/gram dry sediment in lower layer sediments from S0 to S2. The results indicated that anammox bacteria communities might be strongly influenced by mangrove trees. In addition, the correlation analysis showed the redox potential and the molar ratio of ammonium to nitrite in sediments might be important factors affecting the diversity and distribution of anammox bacteria in mangrove sediments.     

Distribution patterns of ammonia-oxidizing bacteria and anammox bacteria in the freshwater marsh of Honghe wetland in Northeast China

Community characteristics of aerobic ammonia-oxidizing bacteria (AOB) and anaerobic ammonium-oxidizing (anammox) bacteria in Honghe freshwater marsh, a Ramsar-designated wetland in Northeast China, were analyzed in this study. Samples were collected from surface and low layers of sediments in the Experimental, Buffer, and Core Zones in the reserve. Community structures of AOB were investigated using both 16S rRNA and amoA (encoding for the α-subunit of the ammonia monooxygenase) genes. Majority of both 16S rRNA and amoA gene-PCR amplified sequences obtained from the samples in the three zones affiliated with Nitrosospira, which agreed with other wetland studies. A relatively high richness of β-AOB amoA gene detected in the freshwater marsh might suggest minimal external pressure was experienced, providing a suitable habitat for β-AOB communities. Anammox bacteria communities were assessed using both 16S rRNA and hzo (encoding for hydrazine oxidoreductase) genes. However, PCR amplification of the hzo gene in all samples failed, suggesting that the utilization of hzo biomarker for detecting anammox bacteria in freshwater marsh might have serious limitations. Results with 16S rRNA gene showed that Candidatus Kuenenia was detected in only the Experimental Zone, whereas Ca. Scalindua including different lineages was observed in both the Buffer and Experimental Zones but not the Core Zone. These results indicated that both AOB and anammox bacteria have specific distribution patterns in the ecosystem corresponding to the extent of anthropogenic impact.     

多形类杆菌的分子生物学鉴定

目的用聚合酶连反应技术(PCR)鉴定多形类杆菌。方法采用16S rRNA基因序列为靶基因设计引物,建立PCR反应体系对该种细菌进行鉴定,并测定PCR产物序列,验证PCR反应的特异性。结果3株多形类杆菌能被扩增,其他菌株和基因组不能被扩增;所测得的PCR产物序列和标准的多形类杆菌16S rRNA基因序列99%同源。结论建立PCR反应体系能特异地鉴定多形类杆菌。     

16S rRNA/ITS基因序列分析与微生物分类鉴定及其在药品微生物质量控制中的应用

经典的微生物鉴定有赖于微生物的纯培养,其在一些难培养、生长缓慢或需要特殊营养的微生物鉴定方面有明显的局限性。全自动细菌鉴定及药敏分析系统和基质辅助激光解离飞行时间质谱对微生物鉴定属表型鉴定,严重依赖仪器生产厂商提供的数据库,对类型多样的环境微生物的鉴定分型有明显缺陷。基于遗传物质的分子生物学鉴定技术为微生物的准确鉴定及分型提供了新的思路。16S rRNA/ITS遗传信息具有相对稳定性和易变异的双重特点,不依赖于微生物的营养及生长状态,在微生物的鉴定、分型中得到广泛应用。微生物是影响药品质量的重要因素,而药品微生物控制是药品质量控制的重要方面。本文对16S rRNA/ITS序列与微生物鉴定、分型及其在药品微生物质量控制领域中的应用进行综述,为相关工作提供参考。     

安罗替尼联合AP方案治疗晚期非小细胞肺癌的临床研究

目的 基于线粒体cytochrome coxidase I（CO I）和16 S rRNA基因开发DNA双重条形码鉴定方法,由此验证并补充形态鉴定,以期建立一种准确、有效地鉴定地龙及混淆品原动物的方法。方法 对收集到的66份样品依据形态特征进行初步鉴定,使用优化后的引物同时扩增CO I和16 S rRNA序列。优化一步法双重PCR实验条件。用MEGA 5.1计算地龙及其混淆品的种内、种间遗传距离,基于K2P模型构建NJ树。结果 CO I和16 S rRNA双重DNA条形码鉴定与形态鉴定结合,可准确鉴别地龙及混淆品原动物。结论 形态鉴定是分子鉴定的基础,分子鉴定是形态鉴定的有力补充。二者结合可最大程度地实现地龙及其混淆品原动物的准确鉴定。DNA双重条形码鉴定方法也可为地龙药材鉴定以及其他动物药材的分子鉴定提供参考。     

Isolation of actinomycetes from the root of the plant, Ophiopogon japonicus, and proposal of two new species, Actinoallomurus liliacearum sp. nov. and Actinoallomurus vinaceus sp. nov

Actinomycete strains K10-0485(T) and K10-0528(T) were isolated from the roots of Ophiopogon japonicus collected in Yokohama, Kanagawa Prefecture, Japan. The 16S ribosomal RNA (rRNA) gene sequences, morphological characteristics and chemotaxonomic data indicated that these strains belonged to the genus Actinoallomurus. Strain K10-0485(T) showed high similarity of the 16S rRNA gene sequence with A. luridus TT02-15(T) (99.1%), but the DNA-DNA hybridization relatedness values between strain K10-0485(T) and A. luridus TT02-15(T) were below 70%. Three species showed similarities of 16S rRNA gene sequences with K10-0528(T), namely A. spadix JCM 3146(T) (98.0%), A. purpureus TTN02-30(T) (98.0%) and A. luridus TT02-15(T) (97.9%), but all similarity values of the 16S rRNA gene sequences were lower than the boundary value (98.7%) for distinguishing as different species. Based on phylogenetic analyses, DNA-DNA hybridization relatedness and physiological characteristics, the two isolated strains should be classified as two new species in the genus Actinoallomurus, and we propose the names Actinoallomurus liliacearum sp. nov. and Actinoallomurus vinaceus sp. nov. The type strain of Actinoallomurus liliacearum is K10-0485(T) (=JCM 17938(T), BCC 49424(T), NBRC 108672(T)) and that of Actinoallomurus vinaceus is K10-0528(T) (=JCM 17939(T), BCC 49425(T), NBRC 108763(T)).