随机扩增多态性DNA技术在鲍氏层孔菌菌株鉴别中的应用

用20个随机引物对7个不同来源的鲍氏层孔菌菌株进行了RAPD分析．结果表明120个随机引物中,有17个引物的扩增产物DNA条带表现出明显的多态性,不同引物对供试菌株扩增出现的DNA条带数目少则10条,多达33条．DNA片段从250bp到2000bp;采用17个引物对7个鲍氏层孔菌菌株共扩增出DNA片段带377条,不同引物扩增出的DNA片段谱带存在较大差异．采用UPGMA系统聚类法,将7个菌株聚类为两大类,能直观准确地揭示菌株间的差异并加以鉴别．     

RAPD方法对甲型溶血性链球菌分型的探讨

甲型溶血性链球菌常居于人类的口腔、＿L呼吸道等部位,为人体的重要条件致病菌。本文应用随机引物PCR或称RAPD法,对甲型溶血性链球菌进行比较,分析该菌RAPD扩增产物的多态性,以判断各菌株间DNA差异和亲缘关系。作者随机设计f17bP和10加的2条引物,用RAPD方法分析了30株甲型溶血性链球菌及5株肺炎链球菌DNAs用17hP引物对30株未卜本地区3所医院临床标本分离的甲型溶血性链球菌DNA进行扩增,共出现了12个DNA片段的条带,分别命名A、B、C……L,各菌株间的主要条带具有较强的相似性。根据A、J2条带分布特征可将30株菌区分为…     

随机扩增多态性DNA分析院内感染的铜绿假单胞菌

目的 运用随机扩增多态性DNA(RAPD)基因分型技术监测铜绿假单胞菌(PA)的医院感染情况,了解耐药表型与RAPD基因型关系,为确定院内交叉感染发生及院感控制提供依据.方法 对10例ICU患者连续多次采样分离出的48株PA进行RAPD基因分型和药物敏感性试验.结果 48株PA共分出9个基因型.6例患者为单一RAPD型PA菌株感染,4例患者检出2种RAPD 型,有3组患者分别检出同型RAPD.耐药性最高的是环丙沙星(75%),其次是左氧氟沙星(73%),耐药性最低的为美洛培南(31%).结论 耐药表型与RAPD基因型之间不存在相关性;RAPD分型快速简单,对于流行病学调查确定院内交叉感染或流行具有重要意义.     

酿酒酵母耐热相关基因DNA片断的初探*

对模板DNAMg^2＋的浓度以及PCR反应程序等因素进行了研究,得出了对酿酒酵母（Saccharomycescerevisiae）进行随机扩增多态性DNA（RAPD）分析的优化条件。在此基础上对酿酒酵母耐高温菌株HU-TY-1及原始出发菌株LK的基因组DNA进行RAPD分析,结果表明：两菌株间确实存在着扩增带型上的差异,而这些差异可能与菌株的耐高温性状有关。从而为今后通过四分子分析寻找与耐热相关基因紧密连锁的分子标记,并进一步克隆和定位有关的基因打下基础。     

应用RAPD标记对斜生褐孔菌菌株亲缘关系的分析

采用RAPD技术对采集不同地区的8个斜生褐孔菌野生菌核分离得到的菌株亲缘关系进行研究,获得了斜生褐孔菌不同菌株的DNA指纹图谱。结果显示:12个引物共扩增出167条带,其中101条为多态性带,多态性比率为60.5%,同一培养时期的各菌株间RAPD图谱表明菌株间存在一定的种内及地理来源差异。若以遗传距离0.508为结合线,可将供试菌株划分为三大类,BCX01、BCX02归为一类,JL01、JL02、JL03、JL04、JL05归为一类,HLJ01单独聚为一类。     

应用RAPD标记对斜生褐孔菌菌株亲缘关系的分析

采用RAPD技术对采集不同地区的8个斜生褐孔菌野生菌核分离得到的菌株亲缘关系进行研究,获得了斜生褐孔菌不同菌株的DNA指纹图谱。结果显示:12个引物共扩增出167条带,其中101条为多态性带,多态性比率为60.5%,同一培养时期的各菌株间RAPD图谱表明菌株间存在一定的种内及地理来源差异。若以遗传距离0.508为结合线,可将供试菌株划分为三大类,BCX01、BCX02归为一类,JL01、JL02、JL03、JL04、JL05归为一类,HLJ01单独聚为一类。     

应用RAPD技术对我国不同地域红色毛癣菌分离株的遗传多样性研究

目的探讨我国不同地域红色毛癣菌分离株的遗传多样性。方法采用随机扩增DNA多态性(RAPD)方法对来源于我国不同地域(江苏南京,山东济南,广东广州)的32株红色毛癣菌临床分离株进行DNA多态性分析。结果红色毛癣菌种内差异明显,根据遗传相似性分成三大聚类群,与地域差异及取材部位无明显相关性,而与表型具有一定相关性。结论随机扩增DNA多态性方法可用于红色毛癣菌的DNA分型,其DNA带型具有一定的遗传变异性,与菌株表型有一定关系,与地域差异、侵犯部位无明显相关性。     

家庭内红色毛癣菌病患者间菌株差异性分析

目的用PCR技术比较分离自同一家庭红色毛癣菌病患者的菌株差异性,分析家庭内多发的红色毛癣菌病的致病菌株是家内相互感染,还是家外感染。方法以家庭内多发的皮肤癣菌病患者为研究对象,分离致病菌株并以传统方法鉴定菌种。再分别用随机扩增多态性DNA(RAPD)和巢式PCR特异扩增红色毛癣菌的串联重复亚元件(TRSS:TRS-1/TRS-2)产生的指纹图谱分析种内株间有无差异性。结果纳入实验的16株菌分离自8个家庭,用形态学等方法及种特异引物均鉴定为红色毛癣菌。RAPD显示4个家庭内的菌株间有差异性,TRS-1区PCR指纹图谱显示5个家庭内菌株有株间差异,TRS-2区能鉴定出2个家庭内菌株间有差异。综合各方法共区分出6个家庭内的菌株间有带型差异。结论该研究提示家庭内多发红色毛癣菌病从家外途径感染率高于家内感染。TRS-1区PCR指纹图谱对红色毛癣菌的菌株区分度高于RAPD,更适于红色毛癣菌株间分型。结合多种分子分型方法可最大限度发现不同菌株间的差异。     

鲍曼不动杆菌随机扩增多态性DNA法基因分型的研究

目的 利用随机扩增多态性DNA(RAPD)技术对鲍曼不动杆菌进行基因分型建立DNA指纹图谱,调查该菌在各临床科室的流行情况,并将其与药敏谱进行比较.方法 随机收集中南大学湘雅二医院2007年9月到2008年9月分离出的86株鲍曼不动杆菌,采用WHO推荐的K-B法对鲍曼不动杆菌进行药物敏感试验,建立药敏谱,同时利用随机扩增多态性DNA法(RAPD)技术进行基因分型,建立DNA指纹图谱,对二者进行比较,然后结合药敏谱和指纹图谱分析各临床科室鲍曼不动杆菌的感染情况.结果 药物敏感试验将86株鲍曼不动杆菌分为47型,RAPD技术将其分为14型,其中A、F、D、B和L型为5种优势型,其菌株数分别为22、15、11、9和7株.本院ICU,老年病科,神经内科,呼吸内科,神经外科鲍曼不动杆菌检出率较高.结论 RAPD分型方法优于药敏分型,其在流行病学研究上更能证实菌株的相关性,在早期发现和预防感染暴发流行中起重要作用.     

三种DNA指纹图谱技术在菌株分型中的应用

旨在通过ERIC-PCR、BOX-PCR、RAPD等技术对4株地芽孢杆菌属菌株进行DNA指纹图谱分析,找到一种适合于地芽孢杆菌属尤其是嗜热脂肪地芽孢杆菌的菌株分型方法。4株地芽孢杆菌属菌株呈现出4种不同的指纹图谱,其中ERIC-PCR的方法获得的条带较为清晰,实验方法较为简便快捷,且能相对较好地区分不同株系;BOX-PCR的方法得到的条带相对较少,不能很好地区分同一种菌的不同株系;RAPD的方法获得的条带相对较多,区分性更高,但同时也增加了一部分工作量和成本。3种菌株分型技术均能有效的区分地芽孢杆菌属菌株,其中ERIC-PCR是一种最有效最便捷区分嗜热脂肪地芽孢杆菌不同株系的方法。     

Qualitative and quantitative characterization of RAPD variation among snap bean (Phaseolus vulgaris) genotypes

Ten snap bean (Phaseolus vulgaris) genotypes were screened for polymorphism with 400 RAPD (random amplified polymorphic DNA) primers. Polymorphic RAPDs were scored and classified into three categories based on ethidium bromide staining intensity. An average of 5.19 RAPD bands were scored per primer for the 364 primers that gave scorable amplification products. An average of 2.15 polymorphic RAPDs were detected per primer. The results show that primer screening may reduce the number of RAPD reactions required for the analysis of genetic relationships among snap-bean genotypes by over 60%. Based on the analysis of the distribution of RAPD amplification, the same number of polymorphic RAPDs were amplified from different genotypes for all RAPD band intensity levels. A comparison of RAPD band amplification frequency among genotypes for the three categories of bands classified by amplification strength revealed a measurable difference in the frequencies of RAPDs classified as faint (weakly amplifying) compared to RAPD bands classified as bold (strongly amplifying) indicating a possible scoring error due to the underscoring of faint bands. Correlation analysis showed that RAPD bands amplified by the same primer are not more closely correlated then RAPD bands amplified by different primers but are more highly correlated then expected by chance. Pairwise comparisons of RAPD bands indicate that the distribution of RAPD amplification among genotypes will be a useful criterion for establishing RAPD band identity. For the average pairwise comparison of genotypes, 50% of primers tested and 15.8% of all scored RAPDs detected polymorphism. Based on RAPD data Nei's average gene diversity at a locus was 0.158 based on all scorable RAPD bands and 0.388 if only polymorphic RAPD loci were considered. RAPD-derived 1 relationships among genotypes are reported for the ten genotypes included in this study. The data presented here demonstrate that many informative, polymorphic RAPDs can be found among snap bean cultivars. These RAPDs may be useful for the unique identification of bean varieties, the organization of bean germplasm, and applications of molecular markers to bean breeding.     

粘类小麦细胞质雄性不育系的RAPD分析

利用250条10-聚寡核苷酸随机引物对具粘果山羊草(Aegilops kotschyi)、易变山羊草(Ae.variabilis)、偏凸山羊草(Ae.ventricosa)和二角山羊草(Ae．bicornis)细胞质不育系及其保持系5-1的总DNA进行了RAPD多态性分析,其中31条引物对4种不育系及其保持系总DNA均无扩增,217条引物扩增条带完全相同。有2条随机引物在2种不育系之间有特异的扩增片段,其中引物S22在偏凸山羊草细胞质雄性不育系基因组DNA中扩增出分子量约为1600bp的特异带,引物S202在粘果山羊草细胞质雄性不育系基因组DNA中扩增出约1300bp特异带。线粒体基因组DNA的RAPD分析表明,4种不育系及其保持系mtDNA存在明显的差异。证明了S22—1600为偏凸山羊草细胞质不育系及其mtDNA基因组DNA的RAPD特异片段．S202—1300可能为粘果山羊草细胞质不育系及其ctDNA基因组DNA的RAPD特异片段。     

Impact of scoring error and reproducibility RAPD data on RAPD based estimates of genetic distance

RAPD band reproducibility and scoring error were evaluated for RAPDs generated by 50 RAPD primers among ten snap bean (Phaseolus vulgaris L.) genotypes. Genetic distances based on different sets of RAPD bands were compared to evaluate the impact of scoring error, reproducibility, and differences in relative amplification strength on the reproducibility of RAPD based genetic distance estimates. The measured RAPD data scoring error was 2%. Reproducibility, expressed as the percentage of RAPD bands scored that are also scored in replicate data, was 76%. The results indicate that the probability of a scored RAPD band being scored in replicate data is strongly dependent on the uniformity of amplification conditions between experiments, as well as the relative amplification strength of the RAPD band. Significant improvement in the reproducibility of scored bands and some reduction in scoring error was achieved by reducing differences in reaction conditions between replicates. Observed primer variability for the reproducibility of scored RAPDs may also facilitate the selection of primers, resulting in dramatic improvements in the reproducibility of RAPD data used in germplasm studies. Variance of genetic distances across replicates due to sampling error was found to be more than six times greater than that due to scoring error for a set of 192 RAPD bands. Genetic distance matrices computed from the RAPD bands scored in replicated data and RAPD bands that failed to be scored in replicated data were not significantly different. Differences in the ethidium bromide staining intensity of RAPD bands were not associated with significant differences in resulting genetic distance matrices. The assumption of sampling error as the only source of error was sufficient to account for the observed variation in genetic distance estimates across independent sets of RAPD bands.     

60Co-γ射线辐射诱变蝴蝶兰M1代花型突变体的RAPD分析

利用72个10bp随机引物对蝴蝶兰（F141780）及其60Co-γ射线辐射的8个表现型花型突变体进行RAPD分析。其中有30个引物能扩增出理想的带型,有4个随机引物扩增出的带型显示其中的25-6、304、30—8-2等3个突变体与对照品种之间具有稳定的多态性差异,即初定其为基因型花型突变体。     

A preliminary study in the use of RAPD markers in detecting genetic differences in hatching patterns of Chirocephalus diaphanus Prévost, 1803 (Crustacea: Anostraca)

Randomly amplified polymorphic DNA (RAPD) was used to detect intra-clutch genetic differences of the anostracan Chirocephalus diaphanus Prévost, 1803. Thirty primers were tested on 130 nauplii from three different clutches. Two primers produced five repeatable and well-defined amplification products, that were used as genotype markers. Nauplii hatched after three successive dehydration and rehydration cycles. Consistent genetic differentiation was observed among nauplii from the same clutch. Significant association between presence or absence of three polymorphic amplification products and hatching after different stimuli was observed. These results suggest that RAPD markers might be related to the difference in nauplii hatching and provide reasonable support to the hypothesis that the source of the observed variation in hatching phenology are genetic as well as epigenetic.     

川西北荞麦种间亲缘关系初步研究

采用RAPD和同工酶技术对川西北具有代表性的10份荞麦材料进行分析.结果表明,筛选出的15个RAPD引物扩增出388条带.其中352条具有多态性,多态性比率为90.72%;过氧化物同工酶分析获得15条酶带,酯酶同工酶获得10条酶带.3种方法聚类结果基本一致,10份材料可初步聚为4个类群.其中,与金荞亲缘关系相比,甜荞较近而苦荞较远;普格县野荞和齿翅野荞、细柄野荞亲缘关系较近,划为同类;阿坝野荞单独划为一类,对其在分类中的地位有待进一步研究.     

小麦双引物RAPD分析方法的研究

ＲＡＰＤ标记是近几年迅速发展的一种新型分子标记,标准的ＲＡＰＤ的反应是以１０个寡聚核苷酸作引物,通过ＰＣＲ反应扩增出基因组的部分片段,我们在研究外源ＤＮＡ导入小麦后外源遗传物质的追踪时,对这个方法进行了改进,采取了双引物进行扩增,结果双引物反应能够比单引物反应扩增出更多的多态性片段。分子杂交结果表明,双引物扩增出的新片段与单引物扩增片段无同源性,并对双引物扩增出的多态性片段产生的可能原因进行讨论。     

Segregation analysis of random amplified polymorphic DNA (RAPD) markers in Picea abies Karst.

The reliability of arbitrarily primed amplification products was tested. The segregation analysis of 266 amplification products obtained using 17 different 10-mer oligonucleotides in 34 megagametophytes from a single tree of Picea abies was carried out. Fifty-four out of the 165 variable bands fit the 1:1 segregation ratio expected for Mendelian traits. The segregation ratio of a subset of six RAPD markers in five other individuals from the same population confirmed their genetic nature. Our results strengthen the evidence previously reported that RAPDs markers can be considered Mendelian traits useful in the detection of genetic variability among both different individuals and populations.     

Survey of Aspergillus species associated with the human respiratory tract

A comprehensive survey has been carried out on the occurrence ofAspergillus species in the respiratory tract of patients of bronchopulmonary diseases in Delhi. In all, 1238 clinical specimens, which included 1082 sputa, 143 bronchial aspirates and 13 throat swabs obtained from 812 patients, were examined. Of these 61.7 per cent patients were culturally positive yielding 29 different species ofAspergillus. The prevalence of aspergilli in sputa was significantly higher than in the bronchial aspirates.Aspergillus niger was the commonest species isolated showing a prevalence of 36.7 per cent. It was followed byA. flavus, A. nidulans, A. terreus, A. versicolor, A. sydowi, A. japonicus andA. oryzae. None of theAspergillus species showed a significant correlation with any of the diseases, or the type of treatment the patients had received. Of the 8 broad occupational groups investigated farmers and labourers showed higher prevalence ofA. niger andA. flavus. The prevalence ofAspergillus species in the throats of healthy persons was 16 per cent withA. versicolor being the commonest species followed byA. flavus, A. amstelodami, A. sydowi andA. terreus. A comparison of the prevalence ofAspergillus species in the patients, healthy individuals and atmosphere of Delhi appears to support the view that the aspergilli are transient residents in the human respiratory tract following their inhalation from the environment.     

Development of reliable PCR-based markers linked to downy mildew resistance genes in lettuce

Summary Sequence characterized amplified regions (SCARs) were derived from eight random amplified polymorphic DNA (RAPD) markers linked to disease resistance genes in lettuce. SCARs are PCR-based markers that represent single, genetically defined loci that are identified by PCR amplification of genomic DNA with pairs of specific oligonucleotide primers; they may contain high-copy, dispersed genomic sequences within the amplified region. Amplified RAPD products were cloned and sequenced. The sequence was used to design 24-mer oligonucleotide primers for each end. All pairs of SCAR primers resulted in the amplification of single major bands the same size as the RAPD fragment cloned. Polymorphism was either retained as the presence or absence of amplification of the band or appeared as length polymorphisms that converted dominant RAPD loci into codominant SCAR markers. This study provided information on the molecular basis of RAPD markers. The amplified fragment contained no obvious repeated sequences beyond the primer sequence. Five out of eight pairs of SCAR primers amplified an alternate allele from both parents of the mapping population; therefore, the original RAPD polymorphism was likely due to mismatch at the primer sites.