利用RAPD技术对木耳属菌株进行分类鉴定的研究

利用ＲＡＰＤ技术对木耳属不同种和种内不同菌株进行了分子鉴定。结果表明,在供试的２６个随机引物中,有１０个引物可扩增出清晰、稳定的ＤＮＡ带型,其中引物Ｓ４和Ｓ６与供试木耳属菌株的基因组ＤＮＡ结合位点少而保守,扩增图谱具有种的特异性,可用于种的快速准确鉴定;其综随机引物的扩增ＤＮＡ多态性较强,可用于种内不同菌株的鉴定;聚类分析表明,在７５％相似水平下,可将供试的木耳属菌株分成四大类,其中有三大类各代表一个形态鉴定的种。研究结果表明了ＲＡＰＤ技术可有效地用于木耳属种或菌株的快速准确鉴定。     

利用RAPD技术对木耳属菌株进行分类鉴定的研究

利用ＲＡＰＤ技术对木耳属不同种和种内不同菌株进行了分子鉴定。结果表明,在供试的２６个随机引物中,有１０个引物可扩增出清晰、稳定的ＤＮＡ带型,其中引物Ｓ４和Ｓ６与供试木耳属菌株的基因组ＤＮＡ结合位点少而保守,扩增图谱具有种的特异性,可用于种的快速准确鉴定;其综随机引物的扩增ＤＮＡ多态性较强,可用于种内不同菌株的鉴定;聚类分析表明,在７５％相似水平下,可将供试的木耳属菌株分成四大类,其中有三大类各代表     

澳大利亚棉花黄萎病菌DNA多态性及其地理分布相关性研究(英文)

应用ＲＡＰＤ技术对澳大利亚东南部八个主要棉花种植区的９９个棉花黄萎病菌菌株进行了ＤＮＡ多态性分析。结果表明用１０个筛选的随机引物对供试菌株的全基因组ＤＮＡ扩增,共获得９２条ＲＡＰＤ谱带,其中５５．４％的谱带为多态带。经类聚分析,供试菌株类聚为１５个ＲＡＰＤ遗传指纹相似组,其中１０个指纹相似组的菌株与其采集区域有明显相关性,其余５个指纹相似组的菌株为普通分布的指纹类型。     

澳大利亚棉花黄萎病菌DNA多态性及其地理分布相关性研究

应用ＲＡＰＤ技术对澳大利亚东南部八个主要棉花种植区的９９个棉花黄萎病菌菌株进行了ＤＮＡ多态性分析。结果表明用１０个筛选的随机引物对供试菌株的全基因组ＤＮＡ扩增,共获得９２条ＲＡＰＤ谱带,其中５５．４％的谱带为多态带。经类聚分析,供试菌株类聚为１５个ＲＡＰＤ遗传指纹相似组,其中１０个指纹相似组的菌株与其采集区域有明显相关性,其余５个指纹相似组的菌株为普通分布的指纹类型。     

多孢节丛孢DNA随机扩增多态性及变种分析

本文对少孢节丛孢的１０个菌株进行了胡机扩增多态性ＤＮＡ（ＲＡＰＤ）分析。４个随机引物获得的ＲＡＰＤ谱带并呈多态性,单个引物获得的ＲＡＰＤ片段数在２～１３个之间。供试的１０个菌株来自不同的地方,形态差异较大,但四个随机引物的扩增结果基本上都体现了少孢节丛孢种的特征“指纹”,基于遗传距离分析和应用ＵＰＧＭＡ方法构建的分子系统树显示,少孢节丛孢不同地理群体间不存在遗传分化,提示在少孢节丛孢群体中没有显著     

烟草黑胫病菌株亲缘关系的RAPD分析

从２２０个ＲＡＰＤ（ＲａｎｄｏｍＡｍｐｌｉｆｉｅｄＰｏｌｙｍｏｒｐｈｉｃ　ＤＮＡｓ）随机引物中所选出的多态扩增性强的２１个引物对来源不同的刀个烟草黑胫病菌株进行全基因组ＤＮＡ遗传多样性分析和指纹构建。选用引物对受试菌株进行ＲＡＰＤ－ＰＣＲ扩增,共产生２４３条ＤＮＡ标记图带,其中１９１条为多态性图带,多态检测率为７８．６％。利用ＵＰＧＭＡ（ＵｎｗｅｉｇｔｈｔｅＰａｉｒ－ｇｒｏｕｐ　ＭｅｔｈｏｄＷｉｔｈＡｒｉｔｈｍｅｔｉｃ　Ａｖｅｒａｇｅ）软件对受试菌株间的遗传距离进行聚类分析构建系统树状图,受试对个菌株被划分为５个遗传聚类组即（Ⅰ、Ⅱ、Ⅲ、Ⅳ、Ⅴ）。结果证明供试菌株具有丰富的遗传多样性,虽各自的遗传背景差异显著,但其亲缘关系相近,遗传聚类组的划分与菌株的地理来源未有明显的相关性。     

RAPD分析快速鉴定双歧杆菌

本文应用ＲＡＰＤ技术,选用１１种引物,以嗜酸乳杆菌为对照,对６种１３珠双歧杆菌菌株基因组ＤＮＡ进行ＰＣＲ扩增,分析其ＤＮＡ指纹图谱,并计算其相似性指数。结果表明,双歧杆菌和非双歧杆菌之间,其相似性指数有显著差异;选择合适的引物进行扩增,双歧杆菌不同种间和同种不同株间可表现不同的ＤＮＡ指纹图谱。本文还对ＲＡＰＤ技术应用于双歧杆菌分类鉴定的可能性进行讨论。     

采用PCR—RFLP和RAPD对球壳孢目真菌系统学的研究

对球壳孢目真菌首次采用ＰＣＲ－ＲＦＬＰ和ＲＡＰＤ进行了系统发育研究,以ＩＴＳ１和ＩＴＳ４为引物对４属１２种２４个菌株的核糖体ＤＮＡ转录间区进行了ＰＣＲ扩增,４种内切酶酶切,结果表明：主要属的ＩＴＳ区长度不同,同属不同种相同。Ｃｏｎｉｏｔｈｙｒｉｕｎ,Ｐｈｙｌｌｏｓｔｉｃｔａ,Ａｓｃｏｃｈｙｔａ,ＳｅｔｏｒｉａＩＴＳ区长度分别为６３０,５６０,５５０,５４０ｂｐ;酶切图各间差别明显,属内种间基本一致     

澳大利亚棉花黄萎病菌DNA多态性及其地理分布相关性研究

应用ＲＡＰＤ技术对澳大利亚东南部八个主要棉花种植区的９９个棉花花萎病菌菌株进行了ＤＮＡ多态性分析。结果表明用１０个筛选的随机引物对供试菌株的全基因组ＤＮＡ扩增,共获得９２条谱带,其中５５．４％的谱带为多态速,经类聚分析,供试菌株类聚为１５个ＲＡＰＤ遗传指纹相似组,其中１０个指纹相似组的菌株与其采集区域有明显相关性,其余５个指纹相似组的菌株为普通分布的指纹类型。     

鹅膏菌属真菌RAPD分析及亲缘关系的研究

本文对采自湖南莽山的２６种鹅膏菌属（Ａｍａｎｉｔａ）真菌进行了随机扩增多态性ＤＮＡ（ＲＡＰＤ）分析,４０个随机引物中筛选出扩增效果较好的６个引物,每个引物能产生１～１０条ＤＮＡ条带,获得的ＲＡＰＤ谱带清晰并呈现多态性ＯＰＧ１５、ＯＰＨ０４两个引物扩增的ＲＡＰＤ谱带能将２６种鹅膏菌完全区分开来,通过６个引物的ＲＡＰＤ分析获得的平均相似性系数表明种与种之间的相关系数在２０－６０％之间,平均链锁聚类分析可将２６种鹅膏菌分为二大类,且一些具菌环和苞状菌托的种类聚在一起,与形态分类基本相吻合。     

冬虫夏草(Cordyceps sinensis)的随机扩增多态DNA及其遗传分化

本文对来自青藏高原3个区域5个具有代表性地方的13个冬虫夏草(Cordyceps sinensis[Berk.] Sacc.)样本进行随机扩增多态DNA(RAPD)分析。19个随机引物获得的RAPD谱带清晰并呈现多态,单个引物获得的RAPD片段数在3～10个之间。该19个引物在每个样本中扩增的RAPD片段总数平均约为65个。基于遗传距离分析,受试的13个冬虫夏草样本中,来自同一地方的样本间遗传差异甚微,同一区域不同地方的样本间遗传差异较大,不同区域的样本间遗传差异最大。这说明冬虫夏草地理群体间存在着遗传分化。应用UPGMA和NJ方法构建的分子系统树显示,来自5个地方的冬虫夏草实际上可以归并为显著不同的3个组,对应于样本来源的3个区域,提示RAPD标记在冬虫夏草群体中有显著的地区特异性。我们的结果还表明,冬虫夏草地理群体间的遗传差异度与地理距离呈正相关。因此,RAPD作为有效的遗传标记,可用于研究冬虫夏草的遗传多样性、起源以及系统演化等。     

A novel specific DNA marker in Saccharomyces bayanus for species identification of the Saccharomyces sensu stricto complex

This study was based on RAPD fingerprinting for species identification of the Saccharomyces sensu stricto complex. 40 random primers were used for RAPD analysis. The results showed that one of these primers, OPT-18, produced a 974 bp species-specific band, which was only found in the tested S. bayanus. Afterward this specific fragment was isolated from agarose gel and ligated into vector for DNA sequencing. A pair of primer SpeOPT18Sbay-F2 and SpeOPT18Sbay-R2 were designed according to the cloned species-specific sequence, which was employed for PCR with the template DNA of the S. sensu stricto strains, single 779 bp species-specific band was only found in S. bayanus. Therefore, we conclude that our novel species DNA marker could be used to rapidly and accurately identify the species of S. bayanus from S. sensu stricto complex by direct PCR.     

穿山甲标本和甲片的DNA提取及PCR扩增

为验证经处理后的穿山甲(Manis spp.)标本和甲片是否可以用于种间分子鉴定标记的开发及个体识别工作,本文在样品的预处理、消化、提取后纯化等方面对传统提取方法进行了改进,分别从穿山甲剥制标本、干皮标本及甲片中提取总DNA;然后用Cyt b基因扩增通用引物、12S rRNA基因全序列扩增引物、RAPD引物及微卫星引物进行了PCR扩增,并对部分扩增结果进行了序列测定.结果表明,除剥制标本的脚底皮张组织外,其他样品基本都可以提取出DNA.以此为模板的PCR扩增中,2种线粒体基因引物扩增出明显目的条带,RAPD引物扩增出种间特异条带,测序结果可用于种间特异性引物及SCAR引物的开发;微卫星引物在甲片样品中扩增稳定,可用于个体识别工作.     

陕西省常见四种实蝇的RAPD研究初报(双翅目:实蝇科)

应用31种随机引物对陕西省常见4种实蝇的基因组DNA进行扩增,筛选出5种引物S61、S107、S126、S275、S1142,可以对4个种扩增出稳定清晰的多态性片段,其中引物S126可以把南瓜实蝇和具条实蝇,具条实蝇和三点棍腹实蝇分开;利用UPGMA法聚类构建的系统树与传统分类完全一致。     

Species specific identification of nine human Bifidobacterium spp. in feces

Based on the 16S rDNA sequences, species specific primers were designed for the rapid identification by DNA amplification of nine human Bifidobacterium spp., namely B. adolescentis, B. angulatum, B. bifidum, B. breve, B. catenulatum, B. dentium, B. infantis, B. longum, B. pseudocatenulatum. B. lactis currently included in dairy products was added to the series. The primers were designed to target different positions of the 16S rDNA, allowing the simultaneous identification of these ten species of Bifidobacterium using two mixtures of primers. The identification procedure described in this paper was validated by establishing a correlation with an AluI restriction pattern of the different full length amplified 16S rDNA. This multiple primer DNA amplification technique was applied for the identification of pure colonies of Bifidobacterium spp. or directly from total bacteria recovered from human fecal samples. The technique was shown to be useful to detect dominant species and, when primers were used in separate reactions, underrepresented species could be identified as well.     

Primers for identification and polymorphism assessment of Vespertilionid bats in the Pacific Northwest

Species‐specific primers for portions of the mitochondrial DNA 16S ribosomal subunit gene were designed to identify bats of the family Vespertilionidae (Mammalia). Two fragments, each of 190 base pairs, were found to contain sufficient genetic variability to consistently resolve 10 of the 14 Pacific Northwest species included in this study. The remaining four species could be resolved into two sets of paired species that were unique from the other 10 species. These primers are demonstrably useful for purposes of surveys designed for species identification of bats, including using DNA extracted from guano collected from roost sites when bats are absent.     

nrDNA ITS和cpDNA rpl16基因测序法对六种鼠尾草的检测

为从鼠尾草属植物中鉴别丹参品种,采用基因测序方法,用核糖体核酸内转录间隔区基因(nrDNA ITS),编码核蛋白体大亚基多肽L16的基因(rpl16)及叶绿体DNA上包含trnL以及trnL和trnF间隔区的区域基因(trnL-trnF)的序列,检测六种鼠尾草属新鲜植物.由于nrDNA ITS和rpl16突变率较高,可以做为6种鼠尾草的基源鉴定标记,依此设计了两对特异引物,从6种鼠尾草中鉴定出丹参(Salvia miltiorrhiza)和云南鼠尾草(S.yunnanensis).但trnL-trnF突变率太低,未能用于鉴别.商品干燥中药材因加工和储藏的方式致使DNA降解严重,基因测序法难于应用.     

扬子鳄鞣制皮革和鳞片的DNA提取方法

运用一种改进的提取方法 ,作者从鞣制皮革中成功地提取了总DNA ,同时还对尾尖皮、鳞片、盐腌生皮等皮质进行了DNA提取 ;用 12SrRNA基因扩增的通用引物、扬子鳄鉴别引物、微卫星引物及RAPD引物进行PCR扩增 ,并对部分扩增结果进行测序 ,以检验提取效果。结果证明 ,几种皮质标本都可提取出DNA ,其中尾尖皮和鳞片的提取效果较好 ,用四种引物都可扩增出明显亮带 ;盐腌生皮和鞣制皮提取结果也很好 ,并且用12SrRNA通用引物、扬子鳄鉴别引物扩增的亮带较明显 ,可进行扬子鳄皮质用品等的分子鉴定及部分序列的扩增和测序研究     

Development of 16S rRNA-gene-targeted group-specific primers for the detection and identification of predominant bacteria in human feces

For the detection and identification of predominant bacteria in human feces, 16S rRNA-gene-targeted group-specific primers for the Bacteroides fragilis group, Bifidobacterium, the Clostridium coccoides group, and Prevotella were designed and evaluated. The specificity of these primers was confirmed by using DNA extracted from 90 species that are commonly found in the human intestinal microflora. The group-specific primers were then used for identification of 300 isolates from feces of six healthy volunteers. The isolates were clearly identified as 117 isolates of the B. fragilis group, 22 isolates of Bifidobacterium, 65 isolates of the C. coccoides group, and 17 isolates of Prevotella, indicating that 74% of the isolates were identified with the four pairs of primers. The remaining 79 isolates were identified by 16S ribosomal DNA sequence analysis and consisted of 40 isolates of Collinsella, 24 isolates of the Clostridium leptum subgroup, and 15 isolates of disparate clusters. In addition, qualitative detection of these bacterial groups was accomplished without cultivation by using DNA extracted from the fecal samples. The goal for this specific PCR technique is to develop a procedure for quantitative detection of these bacterial groups, and a real-time quantitative PCR for detection of Bifidobacterium is now being investigated (T. Requena, J. Burton, T. Matsuki, K. Munro, M. A. Simon, R. Tanaka, K. Watanabe, and G. W. Tannock, Appl. Environ. Microbiol. 68:2420-2427, 2002). Therefore, the approaches used to detect and identify predominant bacteria with the group-specific primers described here should contribute to future studies of the composition and dynamics of the intestinal microflora.