Application of Random Amplified Polymorphic DNA analysis to differentiate strains of Salmonella typhi and other Salmonella species

A Random Amplified Polymorphic DNA (RAPD) fingerprinting method was developed to differentiate isolates of Salmonella serotype typhi ( S. typhi ) and other Salmonella isolates. A panel of five primers was used to examine 63 isolates of Salm. typhi , including 56 strains isolated in Taiwan and seven strains obtained abroad. Twenty-one RAPD types were revealed using the RAPD fingerprinting method. An RAPD with primer 6032 yielded a polymorphism in a 350 bp fragment that differentiated the attenuated vaccine strain Salm. typhi Ty21a from the rest of the Salm. typhi strains. Strains of Salm. typhi were divided into five types with primer D14307. Primer D14307 also proved capable of discrimination among 65 other Salmonella isolates representing 42 different serotypes. The bacterial DNA used in this RAPD protocol was obtained using a commercially available DNA extraction kit (GeneReleaser). The DNA of various strains of Salmonella from this simple extraction procedure could be discriminated within a few hours using the RAPD technique.     

随机扩增多态性DNA技术对结核分支杆菌菌株鉴别及其耐药相关性分析

用引物G1和L1扩增的16S-23SrDNA间隔区作为模板DNA,再用引物AP50进行随机扩增多态性DNA（Random Amplified PolymorphicDNA,RAPD)分棉线AP50扩增出的各菌株DNA片段,用1.2%的琼脂糖凝胶电泳EB染色,可获得1或2个大小不同的DNA片段（390-1031bp)。各株间差异明显,结果表明,不同菌株有不同的DNA多态性,药物敏感株与耐药株之间多态性差异明显,耐药株中耐药谱相似或相同的菌株间其多态性也相似,RAPD有助于简便,快速,有效地了解菌株间的克隆相关性及耐药性传播。     

Random-amplified polymorphic DNA (RAPD) analysis shows intraspecies differences among Xanthomonas albilineans strains

Five strains of Xanthomonas albilineans , causal agent of leaf scald disease in sugarcane from various geographical regions, were compared using random amplification of polymorphic DNA (RAPD) to determine whether they could be differentiated at the DNA level. CsC1-purified genomic DNA from these strains were amplified by the polymerase chain reaction (PCR) using arbitrary 10-mer primers according to standard RAPD conditions and the amplification product profiles analysed by conventional agarose gel electrophoresis. Although most RAPD markers were common to all five strains, unique profiles for each strain were discernible using four 10-mer arbitrary primers individually. Reproducible DNA fingerprints indicate that RAPD analysis can be used to identify and differentiate the X. albilineans strains. This technique has the potential for use in monitoring the appearance of foreign strains of X. albilineans in various geographical regions and could be used for the construction of phylogenetic trees.     

Differentiation of Brucella species by random amplified polymorphic DNA analysis

Random amplification of polymorphic DNA (RAPD) was used for discrimination between 46 Brucella strains and 14 representatives of the alpha-2 and alpha-1 subgroups of Proteobacteria. To evaluate a relatively quick and exact method for Brucella identification, the authors specified the most suitable conditions for RAPD amplification of Brucella DNA with two 10-mer primers, containing lower and higher percentages of G and C. The software package PHYLIP 3.1 was used for cluster analysis of the RAPD fingerprints. The optimization of RAPD conditions resulted in PCR mixes suitable for reliable typing of Brucellae. The distance-based methods (Fitch-Margoliash, UPGMA and Neighbour-joining) gave clear discrimination between Brucella species. The constructed dendrograms put Br. canis and Br. suis bv. 1 in the same cluster and differentiated Brucella strains according to their host preferences. RAPD can be useful method to distinguish related bacterial species, and under strictly established conditions the reaction appears to be a simple, quick and sensitive technique for the epidemiological investigation of brucellosis.     

RAPD (arbitrary primer) PCR is more sensitive than multilocus enzyme electrophoresis for distinguishing related bacterial strains.

The RAPD (random amplified polymorphic DNA) fingerprinting method, which utilizes low stringency PCR amplification with single primers of arbitrary sequence to generate strain-specific arrays of anonymous DNA fragments, was calibrated relative to the widely used, protein-based multilocus enzyme electrophoretic (MLEE) typing method. RAPD fingerprinting was carried out on five isolates from each of 15 major groups of Escherichia coli strains that cause diarrheal disease worldwide (75 isolates in all). Each group consisted of isolates that were not distinguishable from one another by MLEE typing using 20 diagnostic enzyme markers. In our RAPD tests, three or more distinct subgroups in each MLEE group were distinguished with each of five primers, and 74 of the 75 isolates were distinguished when data obtained with five primers were combined. Thus, RAPD typing is far more sensitive than MLEE typing for discriminating among related strains of a species. Despite their different sensitivities, the same general relationships among strains were inferred from MLEE and RAPD data. Thus, our results recommend use of the RAPD method for studies of bacterial population genetic structure and evolution, as well as for epidemiology.     

蚜虫基因组DNA提取方法的改进

蚜虫基因组DNA的提取是蚜虫分子生物学研究中的难点。参照动物基因组DNA的提取方法,根据蚜虫体型微小,体表有外骨骼的特点,对SDS法作了改进。改进的方法无需用组织捣碎棒破碎虫体,操作简便。与现在常用的提取方法相比,改进的SDS法能快速、有效地提取单头蚜虫的基因组DNA,适用于RAPD随机引物和测序引物的PCR扩增。     

人体蠕形螨的DNA提取与随机引物PCR检测

【目的】探索人体毛囊蠕形螨和皮脂蠕形螨DNA的提取方法。【方法】采用液氮反复冻融研磨法破碎螨体细胞, 选用改良小昆虫DNA提取法、碱裂解法和试剂盒提取法, 分别提取冻存时间在5个月内和8~10个月的毛囊蠕形螨和皮脂蠕形螨基因组DNA, 并用随机引物PCR方法进行检测。【结果】蛋白核酸测定仪检测结果显示, 试剂盒法提取的DNA纯度较高、量较多, 明显优于改良小昆虫法和碱裂解法。随机引物扩增结果显示清晰的DNA指纹图谱, 两种人体蠕形螨DNA指纹具有明显差异。蠕形螨冻存时间影响DNA提取的量, 但对DNA提取的纯度和RAPD指纹图谱影响较小。不同DNA提取方法提取的同一种蠕形螨DNA指纹图谱基本相似, 试剂盒法和改良小昆虫法提取的DNA样本条带多而清晰, 碱裂解法提取的样本条带少而模糊。【结论】液氮反复冻融研磨法破碎蠕形螨细胞是有效的, 蠕形螨冻存时间不宜超过6个月, 试剂盒提取法是提取蠕形螨DNA的好方法。RAPD技术可以用于这两种人体蠕形螨DNA分子水平上的检测和分类。     

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.     

Comparison of Haemophilus parasuis reference strains and field isolates by using random amplified polymorphic DNA and protein profiles

ABSTRACT: BACKGROUND: Haemophilus parasuis is the causative agent of Glasser's disease and is a pathogen of swine in high-health status herds. Reports on serotyping of field strains from outbreaks describe that approximately 30% of them are nontypeable and therefore cannot be traced. Molecular typing methods have been used as alternatives to serotyping. This study was done to compare random amplified polymorphic DNA (RAPD) profiles and whole cell protein (WCP) lysate profiles as methods for distinguishing H. parasuis reference strains and field isolates. RESULTS: The DNA and WCP lysate profiles of 15 reference strains and 31 field isolates of H. parasuis were analyzed using the Dice and neighbor joining algorithms. The results revealed unique and reproducible DNA and protein profiles among the reference strains and field isolates studied. Simpson's index of diversity showed significant discrimination between isolates when three 10mer primers were combined for the RAPD method and also when both the RAPD and WCP lysate typing methods were combined. CONCLUSIONS: The RAPD profiles seen among the reference strains and field isolates did not appear to change over time which may reflect a lack of DNA mutations in the genes of the samples. The recent field isolates had different WCP lysate profiles than the reference strains, possibly because the number of passages of the type strains may affect their protein expression.     

Potential of Three-Way Randomly Amplified Polymorphic DNA Analysis as a Typing Method for Twelve Salmonella Serotypes

The potential of a three-way randomly amplified polymorphic DNA (RAPD) procedure (RAPD typing) for typing Salmonella enterica strains assigned to 12 serotypes was analyzed. The series of organisms used included 235 strains (326 isolates) collected mainly from clinical samples in the Principality of Asturias and 9 reference strains. RAPD typing was performed directly with broth cultures of bacteria by using three selected primers and optimized PCR conditions. The profiles obtained with the three primers were used to define RAPD types and to evaluate the procedure as a typing method at the species and serotype levels. The typeability was 100%; the reproducibility and in vitro stability could be considered good. The concordance of RAPD typing methods with serotyping methods was 100%, but some profiles obtained with two of the three primers were obtained with strains assigned to different serotypes. The discrimination index (DI) within the series of organisms was 0.94, and the DI within serotypes Typhimurium, Enteritidis, and Virchow were 0.72, 0.52, and 0.66, respectively. Within these serotypes the most common RAPD types were differentiated into phage types and vice versa; combining the types identified by the two procedures (RAPD typing and phage typing) resulted in further discrimination (DI, 0.96, 0.74, and 0.87, respectively). The efficiency, rapidity, and flexibility of the RAPD typing method support the conclusion that it can be used as a tool for identifying Salmonella organisms and as a typing method that is complementary to serotyping and phage typing methods.     

Potential of three-Way randomly amplified polymorphic DNA analysis as a typing method for twelve Salmonella serotypes.

The potential of a three-way randomly amplified polymorphic DNA (RAPD) procedure (RAPD typing) for typing Salmonella enterica strains assigned to 12 serotypes was analyzed. The series of organisms used included 235 strains (326 isolates) collected mainly from clinical samples in the Principality of Asturias and 9 reference strains. RAPD typing was performed directly with broth cultures of bacteria by using three selected primers and optimized PCR conditions. The profiles obtained with the three primers were used to define RAPD types and to evaluate the procedure as a typing method at the species and serotype levels. The typeability was 100%; the reproducibility and in vitro stability could be considered good. The concordance of RAPD typing methods with serotyping methods was 100%, but some profiles obtained with two of the three primers were obtained with strains assigned to different serotypes. The discrimination index (DI) within the series of organisms was 0.94, and the DI within serotypes Typhimurium, Enteritidis, and Virchow were 0.72, 0.52, and 0.66, respectively. Within these serotypes the most common RAPD types were differentiated into phage types and vice versa; combining the types identified by the two procedures (RAPD typing and phage typing) resulted in further discrimination (DI, 0. 96, 0.74, and 0.87, respectively). The efficiency, rapidity, and flexibility of the RAPD typing method support the conclusion that it can be used as a tool for identifying Salmonella organisms and as a typing method that is complementary to serotyping and phage typing methods.     

Molecular Analysis of the Relatedness of Five Domesticated Turkey Strains

Our knowledge of the genetic relatedness among the eight existing domesticated turkey strains is limited. To begin to address this paucity, genetic relatedness among five turkey strains (Blue Slate, Bourbon Red, Narragansett, Royal Palm, and Spanish Black) was investigated using three molecular marker systems: randomly amplified polymorphic DNA (RAPD), microsatellite, and SNPs derived from a sequence tagged site and a cloned RAPD fragment. The RAPD analyses were based on five primers that revealed a total of 14 informative DNA fragments in all five populations. The microsatellite analyses involved two informative alleles from three primer-pairs. A total of nine SNPs were detected, one of which appeared to be strain specific. This SNP formed the basis of a PCR-RFLP genotyping procedure developed to distinguish one of the strains from the other four. Evidence from these analyses including the SNP-based RFLP-PCR suggests that Royal Palm is distinct from the other four strains, though more closely related to Narragansett. These data provide, for the first time, molecular evidence of the potential relationships among noncommercial domesticated turkey strains.     

Identification of random amplified polymorphic DNA (RAPD) marker for differentiating male from female and sporophytic thalli of <Emphasis Type="Italic">Gracilaria changii</Emphasis> (Rhodophyta)

There have been limited reports on molecular sex markers for macroalgae. We report the use of random amplified polymorphic DNA analysis (RAPD) to identify molecular sex markers for Gracilaria changii (Xia et Abbott) Abbott, Zhang et Xia. Two DNA extraction methods were used: a modified CTAB and phenol-chloroform combination method and the DNeasy Plant Mini Kit. The CTAB and phenol-chloroform method gave the best yield of DNA in quality and quantity and is suitable for larger-sized specimens like G. changii. Sixty-nine RAPD primers were screened to search for sex-linked DNA markers for G. changii, and only one sex-linked marker (716 bp) was identified using OPA 18. RAPD was also used to investigate the molecular characteristics of the three life-stages (male, female, tetrasporophyte) of G. changii. Seven (OPA7, OPA18, S14, S61, S64, S75 and S76) out of the 69 primers showed polymorphism and were selected for interpopulation analysis for DNA isolated from 23 samples collected from Morib and Sungai Pulai in Malaysia. The combination of data produced by the seven primers generated a dendrogram that grouped the specimens into different clades according to their sex and life-stage using the unweighted pair group and arithmetic averages (UPGMA) method. It showed that RAPD was able to differentiate tetrasporophytes, females, and males. Presented at the 6th Meeting of the Asian Pacific Society of Applied Phycology, Manila, Philippines.     

应用RAPD技术鉴别微生态菌种

目的：建立应用RAPD技术鉴别微生态菌种的方法,提高菌种鉴别水平。方法：应用RAPD（随机扩增多态性）方法,选用5条随机引物,利用PCR方法,对3株长双歧杆菌,3株嗜酸乳杆菌,3株粪肠球菌进行基因组DNA指纹图谱分析。结果：发现不同菌株扩增的DNA片段的大小、数量是有差异的。结论：此方法具有可重复性,方便、快速和准确的优势,可用于微生态制剂生产用菌株的鉴别。     

Use of randomly amplified polymorphic DNA as a means of developing genus- and strain-specific Streptomyces DNA probes

We have analyzed 20 randomly amplified polymorphic DNA (RAPD) primers against 36 Streptomyces strains, including 17 taxonomically undefined strains, 25 nonstreptomycete actinomycetes, and 12 outgroups consisting of gram-positive and -negative species. Most of the primers were useful in identifying unique DNA polymorphisms of all strains tested. We have used RAPD techniques to develop a genus-specific probe, one not necessarily targeting the ribosomal gene, for Streptomyces, and a strain-specific probe for the biological control agent Streptomyces lydicus WYEC108. In the course of these investigations, small-scale DNA isolations were also developed for efficiently isolating actinomycete DNA. Various modifications of isolation procedures for soil DNA were compared, and the reliability and specificity of the RAPD methodology were tested by specifically detecting the S. lydicus WYEC108 in DNA isolated from soil.     

Differentiation of <Emphasis Type="Italic">Aspergillus niger</Emphasis> by random amplification of polymorphic DNA

The randomly amplified polymorphic DNA (RAPD) patterns of whole-cell lysates from five Aspergillus niger isolates, including one reference strain, two isolated from deep freeze, and two environmental strains from soil and plant infections, were investigated. PCR-RAPD analysis of genomic DNA was performed using eight primers (Tube-A1, Tube-A6, Tube-A17, Tube-B8, Tube-B11, Tube-B15, Tube-C5, Tube-C6). The RAPD assay discriminated between all strains. Comparison of deep freeze isolates showed identical RAPD patterns in some of the reference and environmental isolates. The data indicates that the RAPD technique is useful for fingerprinting A. niger.     

RAPD analysis of Campylobacter isolates: DNA fingerprinting without the need to purify DNA

A method was developed to obtain reproducible DNA fingerprints from Campylobacter by PCR-based amplification, without the need to isolate total DNA. Randomly amplified polymorphic DNA (RAPD) profiles were generated with three randomly designed 10-mers, using each separately as an amplification primer. A range of C. jejuni serotypes could be typed by RAPD analysis. Depending on the primer, the analysis of RAPD profiles resulted in different levels of discrimination between the strains. Clear correlations were observed between results of RAPD analysis and serotyping. Two of the primers tested generated RAPD profiles which allowed discrimination of strains within given Penner and Lior serotypes.     

大血藤DNA提取及RAPD研究初探

以浙江天台山大血藤为材料,对其DNA提取和RAPD分子标记方法进行了研究。结果表明:所采用的经改进的SDS提取法可获得高质量的大血藤DNA,分子量大于23kb,可满足RPAD扩增;用15个不同的随机引物对所提取的12个个体的大血藤DNA进行了RAPD分子标记分析,其中14个引物扩增产物具有多态性。建立了大血藤DNA提取和RAPD标记的分析程序,为RAPD分析应用于大血藤遗传多样性研究打下了良好的基础。     

Differentiation of Dextran-Producing Leuconostoc Strains by a Modified Randomly Amplified Polymorphic DNA Protocol

Seven dextran-producing Leuconostoc strains were differentiated by using a modified randomly amplified polymorphic DNA (RAPD) protocol that incorporated specific primers designed from conserved regions of dextransucrase genes. RAPD profiles showed intraspecies differences among the Leuconostoc mesenteroides strains tested. This modified RAPD protocol will aid in the differentiation of polymer-producing leuconostocs, which are currently distinguished by time-consuming analyses of the dextrans they synthesize.     

Phylogenetic Analysis of Dipterocarps Using Random Amplified Polymorphic DNA Markers

The phylogenetic relationships among 12 species belonging tothree different genera (Shorea, HopeaandAnisoptera) of Dipterocarpaceaewere studied using random amplified polymorphic DNA (RAPD) markers.A modified CTAB DNA extraction protocol was used to obtain tannin-and polysaccharide-free genomic DNA from mature leaves. Clusteranalysis of data from six random primers placed the 12 speciesin three groups corresponding to their respective genera. Fourdistinct nodes ofShoreaspp. and two ofHopeaspp. could be identified.Anisopteramegistocarpaserved as an outgroup, and was unique when comparedto the other genera examined. RAPD profiles of five individualsofH. odoratawith six random primers were identical, suggestingthat there is little intraspecific variation in this species.The RAPD technique can thus be successfully applied for thestudy of phylogenetic relationships of this important groupof tropical timber trees.Copyright 1998 Annals of Botany Company Dipterocarpaceae,Anisopteraspp.,Hopeaspp.,Shoreaspp., RAPD markers, tropical timber trees.