Random amplified polymorphic DNA analysis of eel genome

Eel family is a huge one, in which many kinds of eels especially some migratory eels, bear strong resemblance to each other, and are therefore difficult to be identified. In this study 29 random primers were used to make RAPD analysis for Japaneses eel (Anguilla japonica), European eel (Anguilla anguilla) and Pike eel (Muraenesox cinereus).And totally 299 fragments were counted.Shared or specific fragments were counted and genetic similarity or genetic distance were calculated.The genetic similarity between Japanese eel and Pike eel is 0.68 and the genetic distance between them is 0.32;those between European eel and Pike eel are 0.72 and 0.28 respectively,and between Japanese eel and European eel are 0.74 and 0.25 respectively.The method has been shown to be suitable to molecular identification of eels.It provides an alternative approach to determine the relationship between species.     

Random amplified polymorphic DNA analysis of genetically modified organisms

Randomly amplified polymorphic DNA (RAPD) was used to analyzed 78 samples comprises of certified reference materials (soya and maize powder), raw seeds (soybean and maize), processed food and animal feed. Combination assay of two arbitrary primers in the RAPD analysis enable to distinguish genetically modified organism (GMO) reference materials from the samples tested. Dendrogram analysis revealed 13 clusters at 45% similarity from the RAPD. RAPD analysis showed that the maize and soybean samples were clustered differently besides the GMO and non-GMO products.     

三种摇蚊幼虫RAPD扩增条件的优化及在昆虫系统发育分析中的应用

为了研究摇蚊科昆虫种群遗传的多样性,以促进对其资源的合理保护,以萨摩亚摇蚊Chironomus samoensisEdwards基因组DNA为模板,对摇蚊幼虫的RAPD扩增条件进行优化,建立了摇蚊幼虫RAPD扩增反应的最佳体系:按照利用优化的RAPD扩增条件进行研究,实验有着良好的重现性。用16个随机引物对3种摇蚊幼虫类群各10个个体进行RAPD扩增,其中萨摩亚摇蚊共扩增出78个条带,多态座位率为41.03%,Shannon遗传多样性指数为0.2570,群体内相似度为0.8730;红裸须摇蚊Propsilocerus akamusi(Tokunaga)共75个条带,多态座位率为44.0%,Shannon遗传多样性指数为0.2472,群体内相似度为0.8731;刺铗长足摇蚊Tanypus punctipennis(Fabricius)共67个条带,多态座位率为41.79%,Shannon遗传多样性指数为0.1943,群体内相似度为0.9066。聚类分析结果表明,刺铗长足摇蚊与红裸须摇蚊的亲缘关系较近。     

Random amplified polymorphic DNA (RAPD) analysis ofPenicillium marneffei strains isolated from AIDS patients in Thailand

Results of random amplified polymorphic DNA (RAPD) analysis using three different primers showed that 16 strains ofPenicillium marneffei isolated from AIDS patients in Thailand belonged to a genetically homogenous group, but different slightly from an isolate from bamboo rat in China. Six PCR fragments (from about, 200 to 600 bp) that were commonly observed in the RAPD fingerprint of all strains were extracted and sequented. Usefulness of this sequence information for identification ofP. marneffei is discussed.     

Random and repetitive primer amplified polymorphic DNA analysis of Bacillus sphaericus

A comparative study of 15 strains representing the five homology groups of Bacillus sphaericus was performed by two PCR methods: RAPD and rep-PCR fingerprinting. The PCR analysis performed with primers corresponding to the naturally occurring repetitive sequences REP, ERIC and BOX, as well as with three random primers, showed highly variable patterns and allowed differentiation of the strains studied. This demonstrated the high discriminative power of the methods. The cluster analysis revealed a low level of similarity between the different homology groups, and within groups I and III, which is evidence of the high genetic heterogeneity of the species B. sphaericus. Close genetic relatedness was observed for the representatives of group IIA, pathogenic to mosquitoes, which supports the idea for differentiation of this group as a separate species.     

Random amplified polymorphic DNA analysis for differentiation of Leuconostoc mesenteroides subspecies isolated from Tenerife cheese

AIMS: In the present study, a RAPD-PCR fingerprinting method was developed to assign Tenerife cheese Leuconostoc mesenteroides strains to its three subspecies (mesenteroides, cremoris and dextranicum). METHODS AND RESULTS: Arbitrarily primed-PCR gave different DNA banding patterns for each type strain of Leuc. mesenteroides subspecies consisting in three major and intense bands of: 1800, 1600 and 1150 bp for subspecies mesenteroides 1800, 1150 and approximately 350 bp for subspecies cremoris; and 1800, 1600 and 1500 bp for subspecies dextranicum. DNA fingerprints of Tenerife cheese Leuc. mesenteroides subspecies were coincident to that of their respective type strain. RAPD profiles were reproducible with DNA template obtained by two different extraction methods. CONCLUSIONS: Tenerife cheese Leuc. mesenteroides strains were rapidly and unequivocally assigned to one of the subspecies by comparing their RAPD-PCR fingerprints with those displayed by type strains used as standards. This technique can be applied to complement preliminary identification of Leuc. mesenteroides to the species level by other molecular methods such as protein fingerprinting. SIGNIFICANCE AND IMPACT OF THE STUDY: RAPD-PCR allows reliable, reproducible and rapid molecular differentiation of Tenerife cheese Leuc. mesenteroides subspecies with no need to use time-consuming and often ambiguous biochemical tests.     

野生稻和栽培稻的随机多态DNA(RAPD)分析

应用 RAPD方法对药用野生稻、普通野生稻、粳稻和籼稻进行基因组多态性分析。 1 2个随机引物共扩增出 1 3 2条 RAPD带 ,片段大小在 3 0 0～ 3 5 0 0 bp之间 ,其中有 1 0 6条表现出多态性 ,占总扩增片段的86.4%。根据遗传距离分析 ,用 UPGMA法构建了聚类树状图 ,结果表明 ,普通野生稻的遗传特性比药用野生稻更接近于栽培稻。     

Random amplified polymorphic DNA analysis and demonstration of genetic variability among bifidobacteria isolated from rats fed with raw kidney beans

A rise in bifidobacterial numbers resembling the Escherichia coli overgrowth phenomenon was observed in the rat small intestine in a feeding experiment with kidney beans. Bifidobacterial colony counts increased from 7.6 x 10(6) to 1.7 x 10(8) cfu.g-1 of intestinal tissue in the anterior part and from less than 1 x 10(5) to 2.65 x 10(8) cfu.g-1 in posterior part of the intestine. Fifteen bifidobacterial strains were purified and further analysed. Random amplified polymorphic DNA (RAPD) assays were used to genetically differentiate bifidobacterial isolates from rat gut and compare them with type strains of 20 different species from the genus Bifidobacterium. A total of 80 arbitrary decamere primers were screened with 6 isolates, and 7 primers were chosen for the final analysis. The amplified DNA bands were scored and analysed by the unweighted pair-group method using arithmetic averages clustering. The isolates were not identical to each other nor to the screened type strains. Whereas it was possible to group 12 of the isolates into 2 separate clusters, 3 strains showed no significant relatedness to any strain. The results of the RAPD analysis indicated that there was a large degree variability among the bifidobacteria in the rat gut and demonstrated the potential applicability of such an approach in the investigation of microbial diversity in complex ecosystems.     

Technical aspects of random amplified polymorphic DNA (RAPD) technique in genotyping of bacterial strains

Random Amplified Polymorphic DNA (RAPD) method became widely applied for sensitive, efficient and fast distinguishing of different isolates of a given species, if pure culture is available. Problems with reproducibility and discriminatory power, frequently cited in the literature, can be overcome by precise optimization procedure allowing to achieve reliable conditions for each species analysed. Basing on two examples of different species, H. pylori and E. faecium, particular parameters of RAPD fingerprinting were evaluated with respect to selection of best working primers generating medium-complex profiles, using only high quality DNA samples and evaluation optimum for every reaction reagent. Stable and informative amplification patterns were obtained with different best working primers which could discriminate between all H. pylori and E. faecium strains tested. For both analysed species different optima were found, suggesting species-specific need of precise RAPD conditions evaluation. This study proved high sensitivity and efficiency of optimized RAPD profiling applicable for searching the epidemiology traces for both species.     

Random amplified polymorphic DNA analysis of Anopheles nuneztovari (Diptera: Culicidae) from Western and northeastern Colombia

Random amplified polymorphic DNA (RAPD) markers were used to analyze 119 DNA samples of three Colombian Anopheles nuneztovari populations to study genetic variation and structure. Genetic diversity, estimated from heterozygosity, averaged 0.34. Genetic flow was greater between the two populations located in Western Colombia (F ST: 0.035; Nm: 6.8) but lower between these two and the northeastern population (F ST: 0.08; Nm: 2.8). According to molecular variance analysis, the genetic distance between populations was significant (phi ST 0.1131, P < 0.001). The variation among individuals within populations (phi ST 0.8869, P < 0.001)was also significant, suggesting a greater degree of population subdivision, not considered in this study. Both the parameters evaluated and the genetic flow suggest that Colombian An. nuneztovari populations are co-specific.     

Isolation and random amplified polymorphic DNA analysis of genomic DNA from soybean embryos

Summary An isolation procedure was developed for the extraction of genomic DNA and Random Amplified Polymorphic DNA (RAPD) analysis using individual soybean embryos. This procedure can be used to quickly and efficiently isolate DNA from a large number of individuals. DNA isolations were analyzed for total yield, integrity, and usefulness as a template in RAPD analysis.     

Hemolysis, toxicity, and randomly amplified polymorphic DNA analysis of Stachybotrys chartarum strains.

Stachybotrys chartarum is an indoor air, toxigenic fungus that has been associated with a number of human and veterinary health problems. Most notable among these has been a cluster of idiopathic pulmonary hemorrhage cases that were observed in the Cleveland, Ohio, area. In this study, 16 strains of S. chartarum isolated from case (n = 8) or control (n = 8) homes in Cleveland and 12 non-Cleveland strains from diverse geographic locations were analyzed for hemolytic activity, conidial toxicity, and randomly amplified polymorphic DNA banding patterns. In tests for hemolytic activity, strains were grown at 23 degrees C on wet wallboard pieces for an 8-week test period. Conidia from these wallboard pieces were subcultured on sheep's blood agar once a week over this period and examined for growth and clearing of the medium at 37 or 23 degrees C. Five of the Cleveland strains (all from case homes) showed hemolytic activity at 37 degrees C throughout the 8-week test compared to 3 of the non-Cleveland strains. Five of the Cleveland strains, compared to two of the non-Cleveland strains, produced highly toxic conidia (>90 microgram of T2 toxin equivalents per g [wet weight] of conidia) after 10 and 30 days of growth on wet wallboard. Only 3 of the 28 strains examined both were consistently hemolytic and produced highly toxic conidia. Each of these strains was isolated from a house in Cleveland where an infant had idiopathic pulmonary hemorrhage.     

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.     

Random amplified polymorphic DNA and restriction enzyme analysis of PCR amplified rDNA in taxonomy: two identification techniques for food-borne yeasts

The random amplified polymorphic DNA (RAPD) assay and the restriction enzyme analysis of PCR amplified rDNA are compared for the identification of the common spoilage yeasts Zygosaccharomyces bailii, Z. rouxii, Saccharomyces cerevisiae, Candida valida and C. lipolytica. Both techniques proved to be adequate tools for yeast identification. Since the RAPD does provide less stable patterns than restriction enzyme analysis of PCR amplified rDNA, and a large amount of data had to be compared without data reduction, Principal Component Analysis (PCA) was applied successfully for clustering the RAPD patterns. The success of PCA is highly influenced by the primer used in RAPD and the amount of reference samples. A large amount of reference samples improves the performance of clustering in PCA. The primer of choice was shown to be important with respect to the discriminatory power of the RAPD method. Some primers used enabled discrimination on the subspecies level. The results collected with both typing methods justify the conclusion that the present typing system can be applied for taxonomical purposes.     

Discrimination of species and strains of basidiomycete genusCoprinus by random amplified polymorphic DNA (RAPD) analysis

All five examined strains ofCoprinus cinereus could be clearly discriminated from the strains of five otherCoprinus species by RAPD patterns with 12 of 13 primers. Also one specimen of unknownCoprinus strain was identified to beC. cinereus by this method. The RAPD patterns were similar among the strains in the same species; many common DNA fragments were recognized as well as some strain-specific DNA fragments. Thus all seven strains ofC. cinereus and all four strains ofC. angulatus examined could be distinguished individually. Diakryotic strains showed the combined RAPD patterns of the two monokaryotic strains constituting the dikaryon. The combined RAPD markers observed in the dikaryons were segregated in their basidiospore progeny. All 18 randomly picked progeny showed different combinations of RAPD markers from the parental strains.     

Plasmid profiles and randomly amplified polymorphic DNA analysis of Salmonella enterica serotype Enteritidis strains from outbreaks and sporadic cases in Turkey

The aim of the study was to investigate the characteristics of Salmonella serotype Enteritidis strains isolated from outbreaks and sporadic cases in Turkey by plasmid profiles and randomly amplified polymorphic DNA (RAPD) patterns. A total of 64 S. Enteritidis clinical strains were selected from the culture collection of the Enterobacteria Laboratory of Ankara University Medical School Department of Microbiology and Clinical Microbiology for molecular analysis using the plasmid profiles and RAPD method. Fifty-six isolates (88%) harbored one to four plasmids ranging in size from 2.5 to 100 kbp. 57 kbp plasmids were the most common plasmids, and forty-four strains (69%) carried 57 kbp plasmids alone or together with other plasmids. The outbreak strains carried the same plasmid profile: three plasmids sized 57, 40, 3.0 kbp. None of the strains analyzed displayed any RAPD bands with the primer OPB-17. By using primer p-1254, 42 strains (66%) were divided into fourteen RAPD patterns. Ten of the outbreak strains (77%) showed >80% similarity by cluster analysis program. Analysis of RAPD-PCR with primer p-1254 proved an easy, rapid and discriminative method complementing antibiogram and plasmid profiles in routine laboratories, and may contribute to the investigations of S. Enteritidis which still cause outbreaks in Turkey. This study presents the first report on S. Enteritidis isolates in Turkey investigated by plasmid profiles and RAPD methods.     

Random amplified polymorphic DNA analysis of DNA extracted from Trichuris trichiura (Linnaeus, 1771) eggs and its prospective application to paleoparasitological studies

Random amplified polymorphic DNA analysis was applied to DNAs extracted from Trichuris trichiura eggs recovered from human fecal samples. Four out of 6 primers tested displayed 18 distinct and well defined polymorphic patterns, ranging from 650 to 3200 base pairs. These results, upon retrieval and DNA sequencing of some of these bands from agarose gels, might help in establishing. T. trichiura specific genetic markers, not available yet, and an important step to design primers to be used in molecular diagnosis approaches.     

Random amplified polymorphic DNA and amplified fragment length polymorphism assessment of genetic variation in Nicaraguan populations of Pinus oocarpa

Pinus oocarpa is the most widely distributed pine species of Mexico and Central America. The natural populations of Nicaragua have been affected by extensive human activities. As a consequence, their size has been reduced, and there is a serious threat to the development of mature woodland. Knowledge of population structures and the genetic diversity of the species is required for the design of sustainable use and conservation strategies. Random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) markers were used to assess the genetic variation among 10 populations from three geographical regions of Nicaragua. Both markers revealed high levels of diversity in these populations. G(ST) values and analyses of molecular variance (AMOVA) found that most variation was within populations but there is still a significant differentiation between populations indicating that the populations sampled cannot be considered a single panmictic unit. The partitions created by AMOVA also showed that there was little differentiation between populations of different regions, although cluster analyses based on RAPDs and AFLPs indicated a closer relationship among most of the populations from a same geographical region. Management of P. oocarpa in Nicaragua should be aimed to maintain the high degree of genetic variation within individual populations that is still observed even in some of these highly degraded populations.     

Differentiation of Lactobacillus sanfranciscensis strains by randomly amplified polymorphic DNA and pulsed-field gel electrophoresis

Genetic diversity of Lactobacillus sanfranciscensis strains isolated from naturally fermented sourdoughs of different origin was evaluated by using randomly amplified polymorphic DNA (RAPD). Computer-assisted comparison of the RAPD patterns revealed a clear separation of L. sanfranciscensis from other obligately heterofermentative Lactobacillus species closely related or normally present in sourdough. Six clusters, five of them constituted by strains of the same origin, were recognized at a similarity level of 63%. Pulsed-field gel electrophoresis (PFGE) results on strains chosen as representative were generally in good agreement with the grouping obtained by RAPD. Both techniques showed a high degree of discriminatory power and indicated the existence of a remarkable genetic polymorphism within the species. Furthermore, the chromosome size of L. sanfranciscensis was estimated by PFGE to be about 1.4 Mb.     

Relationship between biological behaviour and randomly amplified polymorphic DNA profiles of Trypanosoma cruzi strains

Once known some biological characteristics of six Trypanosoma cruzi strains, randomly amplified polymorphic DNA (RAPD) analysis was made. Cluster analysis by UPGMA (unweighted pair group method analysis) was then applied both to biological parameters and RAPD profiles. Inspection of the UPGMA phenograms indicates identical clusters, so supporting that usefulness of biological parameters to characterization of T. cruzi strains still remains.