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.     

Strain typing of Lentinula edodes by random amplified polymorphic DNA assay

Abstract Single 10-base primers were used to generate randomly amplified polymorphic DNA (RAPD) markers in the shiitake mushroom, Lentinula edodes . Seven primers produced polymorphisms in all 15 strains tested, producing 12–19 bands ranging from 0.34 to 2.52 kb. Thirteen of the 15 strains had unique DNA fingerprints, whereas L. edodes ATCC 28759 and ATCC 28760 exhibited identical RAPD profiles for all the primers. Molecular-genetic markers obtained with the RAPD assay can be used to differentiate strains of L. edodes and have potential applications in mushroom breeding and strain improvement programs.     

Identification and typing of food-borne Staphylococcus aureus by PCR-based techniques

The possibility of using PCR for rapid identification of food-borne Staphylococcus aureus isolates was evaluated as an alternative to the API-Staph system. A total of 158 strains, 15 S. aureus, 12 other staphylococcal species, and 131 isolates recovered from 164 food samples were studied. They were phenotypically characterized by API-Staph profiles and tested for PCR amplification with specific primers directed to thermonuclease (nuc) and enterotoxin (sea to see) genes. Disagreement between the PCR results and API-Staph identification was further assessed by the analysis of randomly amplified polymorphic DNA (RAPD) profiles obtained with three universal primers (M13, T3, and T7) and 16S rDNA sequencing. Forty out of 131 isolates (31%) tested positive for PCR enterotoxin. Of these, 14 (11%) were positive for sea, 22 (17%) for sec, one (0.8%) for sed, and three (2.2%) for sea and sec. No amplification corresponding to seb nor see was obtained. Cluster analysis based on RAPD profiles revealed that most of the sec positive food isolates grouped together in three clusters. Cluster analysis combining the three RAPD fingerprints (M 13, T3, and T7), PCR-enterotoxin genotype and API-Staph profiles, grouped the nuc PCR positive isolates together with S. aureus reference strains and the nuc PCR negative isolates with reference strains of other staphylococcal species. The only nuc PCR positive food isolate that remained unclustered was a sed positive strain identified by 16S rDNA sequence as S. simulans. The high concordance between S. aureus and nuc PCR positive strains (99%) corroborates the specificity of the primers used and the suitability of nuc PCR for rapid identification of S. aureus in routine food analysis.     

Characterization of beta-lactam-resistant Bacteroides fragilis isolates by use of PCR fingerprinting

PCR fingerprinting was used for characterization of 35 beta-lactam-resistant Bacteroides fragilis strains isolated in Sweden and Hungary. Ten B. fragilis strains showed unique PCR fingerprints by use of the M13 core primer. Their main product was a DNA fragment with a length of 2000-bp which was absent in the other 25 strains and the reference strain B. fragilis ATCC 25285. The 2000-bp fragment from four imipenem-resistant strains gave rise to positive reactions in a specific PCR for detection of ccrA. Printed by the T3B primer, five B. fragilis strains, including the imipenem-resistant strains showed unique PCR fingerprints. The investigated imipenem-resistant strains produced carbapenem-hydrolysing metallo-beta-lactamases. The study indicates that the unique PCR fingerprinting profiles shown in highly beta-lactam resistant B. fragilis strains are correlated to antimicrobial resistance. The PCR fingerprinting technique is a useful tool for differentiation of Bacteroides fragilis strains with high-level beta-lactam resistance.     

Detection of Polymorphic DNA and Taxonomic Relationships Among 10 Wild Perennial Soybean Species Using Specific and Arbitrary Nucleotide Primers

Polymorphic DNA in complex genomes of agronomic crops can be detected using specific nucleotide and arbitrary primers and the polymerase chain reaction (PCR). Nineteen accessions representing 10 species of the wild perennial soybean were evaluated using 4 sets of specific primers and 3 sets of random amplified polymorphic DNAs (RAPD) primers. The potential of the RAPD assays was further increased by combining two primers in a single PCR. The fragments generated by the two assays discriminated 10 wild species by banding profiles. The size of the amplified DNA fragments ranged from 100 to 2100 base pairs. The resolved PCR products yielded highly characteristic and homogeneous DNA fingerprints. The fingerprints were useful not only for investigating genetic variability but also for further characterizing the wild soybean species by detecting inter- and intra-specific polymorphisms, constructing dendrograms defining the phylogenetic relationships among these species, and identifying molecular markers for the construction of genetic linkage maps. Furthermore, unique markers distinguishing particular species were also identified. Thus, it is expected that PCR will have great relevance for taxonomic studies. This revised version was published online in July 2006 with corrections to the Cover Date.     

Occurrence and community composition of fast-growing Mycobacterium in soils contaminated with polycyclic aromatic hydrocarbons

Fast-growing mycobacteria are considered essential members of the polycyclic aromatic hydrocarbons (PAH) degrading bacterial community in PAH-contaminated soils. To study the natural role and diversity of the Mycobacterium community in contaminated soils, a culture-independent fingerprinting method based on PCR combined with denaturing gradient gel electrophoresis (DGGE) was developed. New PCR primers were selected which specifically targeted the 16S rRNA genes of fast-growing mycobacteria, and single-band DGGE profiles of amplicons were obtained for most Mycobacterium strains tested. Strains belonging to the same species revealed identical DGGE fingerprints, and in most cases, but not all, these fingerprints were typical for one species, allowing partial differentiation between species in a Mycobacterium community. Mycobacterium strains inoculated in soil were detected with a detection limit of 10(6) CFU g(-1) of soil using the new primer set as such, or approximately 10(2) CFU g(-1) in a nested PCR approach combining eubacterial and the Mycobacterium specific primers. Using the PCR-DGGE method, different species could be individually recognized in a mixed Mycobacterium community. This approach was used to rapidly assess the Mycobacterium community structure of several PAH-contaminated soils of diverse origin with different overall contamination profiles, pollution concentrations and chemical-physical soil characteristics. In the non-contaminated soil, most of the recovered 16SrRNA gene sequence did not match with previous described PAH-degrading Mycobacterium strains. In most PAH-contaminated soils, mycobacteria were detected which were closely related to fast-growing species such as Mycobacterium frederiksbergense and Mycobacterium austroafricanum, species that are known to include strains with PAH-degrading capacities. Interestingly, 16S rRNA genes related to M. tusciae sequences, a Mycobacterium species so far not reported in relation to biodegradation of PAHs, were detected in all contaminated soils.     

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.     

Simultaneous molecular subtyping and shiga toxin gene detection in Escherichia coli using multiplex polymerase chain reaction

A robust random amplification of polymorphic DNA (RAPD)-polymerase chain reaction (PCR) protocol was developed for the combined epidemiological typing and shiga toxin detection of clinical shiga toxin-producing O157 and non-O157 Escherichia coli isolates. Using shiga toxin gene-specific primers, combined with two short 10-mer primers, in a multiplex shiga toxin/RAPD-PCR the fingerprints generated allowed differentiation between epidemiologically unrelated strains and allowed identification of a band amplified from the shiga toxin gene(s). Hybridization with a digoxigenin-labelled probe specific for stx1 and stx2 confirmed its identity. The combination of primers in this way allows valuable additional information to be gained from discriminatory RAPD profiles, with further benefits of time and cost savings over tests performed individually.     

Genomic fingerprinting using arbitrarily primed PCR and a matrix of pairwise combinations of primers.

Polymorphisms in genomic fingerprints generated by arbitrarily primed PCR (AP-PCR) can distinguish between slightly divergent strains of any organism. Single oligodeoxyribonucleotide (oligo) primers have been used to generate such fingerprints, with the same primer being present at the 5' end of both strands for every PCR product. We used three arbitrary oligos, individually and in pairs, to generate six different genomic fingerprints of the same mouse genomic DNAs. Fewer than half of the products in genomic fingerprints generated using the oligos in pairs were the same as those produced by AP-PCR using one of the three oligos alone. Thus, a few oligos could be used in a very large number of single and pairwise combinations, each producing a distinct AP-PCR fingerprint with the potential to identify new polymorphisms. For example, 50 oligos can be used in a matrix of pairwise combinations to produce 2,500 fingerprints, in which at least half the data can be expected to be unique to each pair. We demonstrate this principle by using two oligos, alone and together, to generate three sets of fingerprints and map thirteen polymorphisms in the C57BL/6J x DBA/2J set of recombinant inbred mice.     

Genetic and phenetic analyses of Bradyrhizobium strains nodulating peanut (Arachis hypogaea L.) roots.

Seventeen Bradyrhizobium sp. strains and one Azorhizobium strain were compared on the basis of five genetic and phenetic features: (i) partial sequence analyses of the 16S rRNA gene (rDNA), (ii) randomly amplified DNA polymorphisms (RAPD) using three oligonucleotide primers, (iii) total cellular protein profiles, (iv) utilization of 21 aliphatic and 22 aromatic substrates, and (v) intrinsic resistances to seven antibiotics. Partial 16S rDNA analysis revealed the presence of only two rDNA homology (i.e., identity) groups among the 17 Bradyrhizobium strains. The partial 16S rDNA sequences of Bradyrhizobium sp. strains form a tight similarity (> 95%) cluster with Rhodopseudomonas palustris, Nitrobacter species, Afipia species, and Blastobacter denitrificans but were less similar to other members of the alpha-Proteobacteria, including other members of the Rhizobiaceae family. Clustering the Bradyrhizobium sp. strains for their RAPD profiles, protein profiles, and substrate utilization data revealed more diversity than rDNA analysis. Intrinsic antibiotic resistance yielded strain-specific patterns that could not be clustered. High rDNA similarity appeared to be a prerequisite, but it did not necessarily lead to high similarity values between RAPD profiles, protein profiles, and substrate utilization. The various relationship structures, coming forth from each of the studied features, had low compatibilities, casting doubt on the usefulness of a polyphasic approach in rhizobial taxonomy.     

Amplification fragment length polymorphism in Brucella strains by use of polymerase chain reaction with arbitrary primers.

DNA heterogeneity among members of the genus Brucella was demonstrated with the arbitrarily primed polymerase chain reaction (AP-PCR). Simple, reproducible genomic fingerprints from DNA of 25 different Brucella strains were generated with five arbitrarily chosen primers, alone and in pairs, with the PCR. Reaction conditions were optimized for each primer. Several DNA segments were amplified in each sample with all of the primers. PCR products that are not shared among all strains act as polymorphic markers. Polymorphism was apparent for each primer. The Brucella strains can be distinguished according to the banding patterns of their amplified DNA on agarose gels, and the differences can be diagnostic of specific strains. To determine genetic relatedness among the Brucella strains, similarity coefficients were calculated. Statistical analysis of the similarity coefficients revealed the degrees of relatedness among strains of the genus Brucella.     

Intraspecies variability of Desulfovibrio desulfuricans strains determined by the genetic profiles

Fifteen (soil and intestinal) strains of Desulfovibrio desulfuricans species were typed by PCR method with the use of primers specific for repetitive extragenic palindromic (REP) and enterobacterial repetitive intergenic consensus (ERIC) sequences. As a result, characteristic DNA fingerprints for the strains were obtained. Moreover, the genetic profiles were found to be useful for typing and distinguishing the strains of D. desulfuricans. According to cluster analysis, PCR with primers complementary to the sequences REP appeared to be slightly more discriminatory than PCR with ERIC primers for the investigated strains. Distinct fingerprint patterns of two isolates derived from the same patient pointed to the different origin of both strains.     

Genetic Fingerprinting of Pseudomonas syringae Pathovars Using ERIC-, REP-, and IS50-PCR

PCR fingerprinting using primers corresponding to repetitive (ERIC and REP) and insertion sequences (IS50) was investigated as a method to distinguish the pathovars of Pseudomonas syringae . After amplification of total DNA with the ERIC-, REP-, and IS50-PCR followed by agarose gel electrophoresis. most of the tested pathovars showed specific patterns of PCR products. The differences between the fingerprints among strains within a pathovar were small, with the exception of pathovars syringae, aptata , and atrofaciens . The fingerprints of the related pathovars savastanoi, phaseolicola, glycinea, morsprunorum, tabaci, lachrymans , and mori generated with the ERIC- and REP-primers were found to be very similar, showing the potential of this technique for taxonomical studies. In contrast, the IS50-PCR fingerprints of these pathovars were clearly distinguishable. The fingerprint patterns of a strain were highly reproducible with all three tested primer sets, also when whole cells were added to the reaction mixture. Thus, the PCR technique with the ERIC-, REP-, and IS50-primers is a rapid, simple, reproducible, and low cost method to identify and classify strains of the Pseudomonas syringae pathovars.     

Analysis of major band of Enterobacter sakazakii by ERIC-PCR and development of a species-specific PCR for detection of Ent. sakazakii in dry food samples

ERIC (Enterobacterial Repetitive Intergenic Consensus)-PCR was employed to generate stable and reproductive ERIC-PCR fingerprints of Ent. sakazakii ATCC51329. Moreover, this study also cloned and sequenced a major band of Ent. sakazakii (ATCC51329) ERIC-PCR fingerprints. The major band was amplified with primer ERIC2 and sequences extending primer ERIC 2 showed poor similarity with ERIC elements. A comparison of the nucleotide acid with other sequences available in the GenBank revealed 90% of identity with Ent. sakazakii ATCC BAA-894, and 73%-74% of identity with oligopeptiase gene or protease gene of some species from the Enterobacteriaceae family. Two primers were synthesized to develop and optimize an Enterobacter sakazakii-specific PCR based on regions of major band unique to Ent. sakazakii. The expected fragment was amplified from all of Ent. sakazkaii but not from the negative controls. As few as 10(2) CFU/ml of Ent. sakazakii of PCR were directly detected in the infant formulas. This was the case even in the presence of other bacteria. A comparison of traditional methods and new developed PCR in commercial foods suggested that without using API20-E test, the DFI chromogenic medium and FDA method showed 46.15% and 50% false positive respectively. Moreover, one false negative was observed with FDA method. In contrast, PCR was highly sensitive and specific to Ent. sakazakii. A high heterogeneity between Ent. sakazakii and the other microorganisms was found on expected fragment sequence. In addition, Ent. sakazakii ATCC51329 formed a separate branch with >5% divergence from the type strain ATCC BAA-894 and major strains.     

Utilisation of enterobacterial repetitive intergenic consensus (ERIC) sequence-based PCR to fingerprint the genomes of Bifidobacterium isolates and other probiotic bacteria

Enterobacterial repetitive intergenic consensus based on PCR (ERIC-PCR) was used to generate DNA fingerprints for bifidobacteria and other probiotic bacteria. Two primers (ERIC 1R and ERIC 2) used in ERIC-PCR revealed that all of the probiotic bacteria tested possess enterobacterial repetitive intergenic consensus sequences with the PCR products ranging from 250 bp to 5000 bp. The bacterial strains can be differentiated by comparing fingerprint patterns. The dendrogram of the fingerprints revealed that most of the bifidobacterial wild type strains fell into one cluster at similarity level of approximately 79%.     

Characterization of Bilophila wadsworthia Isolates Using PCR Fingerprinting

Bilophila wadsworthia, an under-appreciated anaerobic organism, was originally described in 1989. Ninety-nine Bilophila wadsworthia isolates, recovered form environmental and clinical specimens in Germany and in Southern California, were examined in this study. Many isolates were recovered in mixed culture with facultative aerobic and other anaerobic bacteria. All isolates were identified by standard laboratory procedures, including gas–liquid chromatography (GLC). A PCR fingerprint assay was established to compare the profiles of clinical and environmental isolates to the type strain (ATCC 49260) and to an environmental (sewage) reference strain (DSM 11045, RZATAU) for intra-species differences. Two primers, one universal primer, M13 core, and one tDNA primer, T3B, were used individually to analyse the strains. Homogeneous PCR fingerprint profiles were found for the majority of strains using the M13 core primer; two PCR groups were determined with T3B, one matching the type strain and one matching the environmental reference strain (DSM 11045, RZATAU). Two urease negative strains, WAL 11470 (blood isolate from California) and TÜB 754 (intra-abdominal isolate from Germany) formed unique PCR fingerprint profiles with each of these primers. These results were confirmed by PCR fingerprinting using the T3A primer. These latter results suggest a possible genetic diversity in B. wadsworthia.     

Occurrence and phylogenetic diversity of Sphingomonas strains in soils contaminated with polycyclic aromatic hydrocarbons

Bacterial strains of the genus Sphingomonas are often isolated from contaminated soils for their ability to use polycyclic aromatic hydrocarbons (PAH) as the sole source of carbon and energy. The direct detection of Sphingomonas strains in contaminated soils, either indigenous or inoculated, is, as such, of interest for bioremediation purposes. In this study, a culture-independent PCR-based detection method using specific primers targeting the Sphingomonas 16S rRNA gene combined with denaturing gradient gel electrophoresis (DGGE) was developed to assess Sphingomonas diversity in PAH-contaminated soils. PCR using the new primer pair on a set of template DNAs of different bacterial genera showed that the method was selective for bacteria belonging to the family Sphingomonadaceae.Single-band DGGE profiles were obtained for most Sphingomonas strains tested. Strains belonging to the same species had identical DGGE fingerprints, and in most cases, these fingerprints were typical for one species. Inoculated strains could be detected at a cell concentration of 10(4) CFU g of soil(-1). The analysis of Sphingomonas population structures of several PAH-contaminated soils by the new PCR-DGGE method revealed that soils containing the highest phenanthrene concentrations showed the lowest Sphingomonas diversity. Sequence analysis of cloned PCR products amplified from soil DNA revealed new 16S rRNA gene Sphingomonas sequences significantly different from sequences from known cultivated isolates (i.e., sequences from environmental clones grouped phylogenetically with other environmental clone sequences available on the web and that possibly originated from several potential new species). In conclusion, the newly designed Sphingomonas-specific PCR-DGGE detection technique successfully analyzed the Sphingomonas communities from polluted soils at the species level and revealed different Sphingomonas members not previously detected by culture-dependent detection techniques.     

Fingerprinting genomes using PCR with arbitrary primers.

Simple and reproducible fingerprints of complex genomes can be generated using single arbitrarily chosen primers and the polymerase chain reaction (PCR). No prior sequence information is required. The method, arbitrarily primed PCR (AP-PCR), involves two cycles of low stringency amplification followed by PCR at higher stringency. We show that strains can be distinguished by comparing polymorphisms in genomic fingerprints. The generality of the method is demonstrated by application to twenty four strains from five species of Staphylococcus, eleven strains of Streptococcus pyogenes and three varieties of Oryza sativa (rice).     

Development of strain-specific PCR primers based on a DNA probe Fu12 for the identification of fusobacterium nucleatum subsp. nucleatum ATCC 25586T

The objective of this study was to assess the strain-specificity of a DNA probe, Fu12, for Fusobacterium nucleatum subsp. nucleatum ATCC 25586T (F. nucleatum ATCC 25586T), and to develop sets of strain-specific polymerase chain reaction (PCR) primers. Strain-specificity was tested against 16 strains of F. nucleatum and 3 strains of distinct Fusobacterium species. Southern blot hybridization revealed that the Fu12 reacted exclusively with the HindIII-digested genomic DNA of F. nucleatum ATCC 25586T. The results of PCR revealed that three pairs of PCR primers, based on the nucleotide sequence of Fu12, generated the strain-specific amplicons from F. nucleatum ATCC 25586T. These results suggest that the DNA probe Fu12 and the three pairs of PCR primers could be useful in the identification of F. nucleatum ATCC 25586T, especially with regard to the determination of the authenticity of the strain.     

Identification and Phylogenetic Analysis of Toxigenic Cyanobacteria by Multiplex Randomly Amplified Polymorphic DNA PCR

Randomly amplified polymorphic DNA PCR was used to generate unique and identifying DNA profiles for members of the cyanobacterial genera Anabaena and Microcystis, which are responsible for much of the production of nuisance blooms in various freshwater systems, including recreational and drinking water supplies. A method based on the combination of two 10-mer oligonucleotides in a single PCR was developed to provide specific and repeatable DNA fingerprints for cyanobacterial isolates. The strain-specific randomly amplified polymorphic DNA profiles made it possible to discriminate among all toxigenic cyanobacteria studied to the three taxonomic levels of genus, species, and strain. Analysis of DNA typing results obtained by the described method clearly distinguishes between the genera Anabaena and Microcystis. The markers produced for each strain were also applied to a phylogenetic analysis to infer genetic relatedness in this group of prokaryotes.