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PubMed1997

Use of polymerase chain reaction fingerprinting to compare clinical isolates of Bacteroides fragilis and Bacteroides thetaiotaomicron from Germany and the United States.

Accurate identification of Bacteroides species is often problematic. Therefore, we used a polymerase chain reaction (PCR) fingerprinting technique with either a single nonspecific primer derived from tDNA intergenic spacer or a single primer that anneals to mini- and microsatellite DNA sequences to compare 34 clinical isolates of B. fragilis and 21 clinical isolates of B. thetaiotaomicron from Southern California with 32 clinical isolates of B. fragilis and 10 isolates of B. thetaiotaomicron from Germany. All German B. fragilis isolates (32 of 32) formed one PCR fingerprint group that matched the PCR profile of the B, fragilis reference strain ATCC (American Type Culture Collection) 25285, representative of DNA homology group I. In contrast, the isolates from Southern California formed two PCR fingerprint groups. Although most of these strains (29 of 34) also matched B. fragilis ATCC 25285, some strains (4 of 34) matched the DNA homology group II reference strain VPI (Virginia Polytechnic Institute) 2393. One of the 34 strains showed a unique profile. German B. thetaiotaomicron strains (10 of 10) formed one PCR fingerprint group, matching the reference strain B. thetaiotaomicron ATCC 29742, whereas the B. thetaiotaomicron isolates from Southern California showed heterogenous profiles.

Bacteroides↗
PubMed1997

Bilophila wadsworthia clinical isolates compared by polymerase chain reaction fingerprinting.

Bilophila wadsworthia isolates recovered from a right-ear cholesteatoma and brain abscess of the same patient were analyzed by means of polymerase chain reaction (PCR) fingerprinting with single primers (T3B and M13 core) to ascertain if they originated from the same clone. Their PCR fingerprint profiles were compared with those of three additional B. wadsworthia clinical isolates and the type strain (ATCC 49260). The two isolates from the same patient produced PCR fingerprint profiles identical to each other, regardless of which primer was used. All isolates' PCR fingerprint profiles, with use of either the T3B or M13 core primer, shared some major and minor bands. However, differences in additional major and minor bands distinguished each of the additional isolates, suggesting that there are different subgroups of B. wadsworthia.

Brain Abscess↗
PubMed2004

Automated ordering of fingerprinted clones.

MOTIVATION: A considerable amount of human intervention is currently required to produce high-quality fingerprint-based physical maps for genomic studies. RESULTS: An algorithm has been developed and implemented to automatically order fingerprinted clones within contigs. The resulting software, named CORAL (Clone ORdering ALgorithm), has been tested on maps that have previously been manually edited and on maps derived from in silico simulations. The fingerprint map and DNA sequence of the human genome has provided an additional test to CORAL. Measurements suggest that CORAL performs significantly better than the software currently used by most laboratories to order fingerprinted clones at throughputs far exceeding those that can be achieved manually.

Algorithms↗
PubMed1996

Characterization of indole-negative Bacteroides fragilis group species with use of polymerase chain reaction fingerprinting and resistance profiles.

Biochemical tests alone do not adequately differentiate the various Bacteroides species, groups, and antimicrobial-resistant variants. Consequently, we used a polymerase chain reaction (PCR) fingerprinting technique, with either a single nonspecific primer derived from the t-DNA intergenic spacer region (T3B) or a single primer that anneals to minisatellite DNA sequences (M13 core), to identify and characterize 58 clinical isolates of Bacteroides fragilis group species (B. fragilis, B. distasonis, and B. caccae). In addition to species- and subspecies-specific differences, 4 strains of B. fragilis, 1 of B. distasonis, and 3 of B. caccae that showed increased resistance to imipenem, ampicillin, and ampicillin/sulbactam also produced unique PCR fingerprint profiles. Analysis by the clinical source of isolation (i.e. blood or intraabdominal, skin, or soft-tissue infection) indicated that no particular PCR fingerprint type was associated with greater pathogenicity of any individual clinical source. The PCR fingerprinting technique proves to be a useful tool for species identification and taxonomic studies, as well as for epidemiological studies of Bacteroides species.

Bacterial Typing Techniques↗
PubMed1997

Retrospective detection of laboratory cross-contamination of Mycobacterium tuberculosis cultures with use of DNA fingerprint analysis.

In 1992-1993, we investigated possible cross-contamination of Mycobacterium tuberculosis cultures as part of a study of tuberculosis in Arkansas by using DNA fingerprint analysis. Of patients whose isolates were matched, those for whom smears were negative and only one culture was positive were identified from laboratory records. Clinical, laboratory, DNA fingerprint, and epidemiological data were reviewed. Of 259 patients, nine (3.5%) were judged to be due to cross-contamination. None of these patients had a clinical course consistent with tuberculosis. All nine specimens were processed with another isolate with a matching DNA fingerprint, and epidemiological connections were not identified among any of the patients. To avoid erroneous diagnoses and unnecessary therapy and public health investigations, specimens from patients with tuberculosis whose smears are negative and only one culture is positive should be investigated for cross-contamination. An inconsistent clinical course and a DNA fingerprint that matches those of other culture-positive specimens processed concurrently, coupled with the lack of an epidemiological connection, suggest cross-contamination.

Adult↗
PubMed1996

A comparative study of different PCR-based DNA fingerprinting techniques for typing of the Acinetobacter calcoaceticus-A. baumannii complex.

Different PCR-based DNA fingerprinting techniques were evaluated for typing 26 clinical isolates belonging to the Acinetobacter calcoaceticus-A. baumannii complex. Seven isolates belonged to a previously defined outbreak while 19 isolates were unrelated epidemiologically. The PCR-based DNA fingerprinting techniques used were: (i) repetitive extragenic palindromic (REP) PCR; (ii) enterobacterial repetitive intergenic consensus (ERIC) PCR; (iii) randomly amplified polymorphic DNA with M13 forward primer; (iv) restriction analysis of the amplified 16S rRNA gene (ARDRA-16S); and (v) restriction analysis of an amplified region containing the 16S-23S rRNA spacer region and part of the 23S rRNA gene (ARDRA 23S + spacer). The discrimination index for the PCR-based DNA fingerprinting techniques was: 0.99 for REP; 0.94 for ERIC; 0.87 for M13; 0.60 for ARDRA-16S digested with Hpa II and <0.50 for ARDRA 23S + spacer. It was concluded that REP-PCR possessed high discriminatory power and reproducibility in comparison with the other PCR-based DNA fingerprinting techniques, and is a simple and rapid typing method for use in epidemiological studies of isolates belonging to the A. calcoaceticus-A. baumannii complex.

Acinetobacter↗
PubMed1997

Comparison of Vibrio cholerae O1 isolates by polymerase chain reaction fingerprinting and ribotyping.

The rRNA gene restriction patterns and the polymerase chain reaction (PCR) fingerprinting types of 53 Vibrio cholerae O1 isolates were studied. Five and eight patterns were observed from 27 toxigenic and 26 non-toxigenic O1 isolates after BglI cleavage. PCR fingerprinting with three primer sets aimed at enterobacterial repetitive intergenic consensus (ERIC) sequences, ERIC-related sequences in V. cholerae, another kind of repeated sequences in V. cholerae (VCR) and arbitrary sequences divided the same strains into seven and 10 PCR types, respectively. Eight ribotypes had unique PCR patterns. PCR fingerprinting identified more than one pattern among isolates within each of the remaining ribotypes. However, ribotyping was able to differentiate the same PCR types in one case. A single ribotype and a single PCR pattern were found in toxigenic O1 strains isolated in Taiwan from imported food and imported cases of cholera between 1993 and 1995. Typing of V. cholerae O1 by PCR fingerprinting correlated well with ribotyping, but was more discriminating. PCR assay provides a rapid and simple means of typing these strains for epidemiological studies.

Cholera↗
PubMed1997

Direct comparison of two commercially available computer programs for analysing DNA fingerprinting gels.

Randomly amplified polymorphic DNA (RAPD) fingerprints were generated with M13 and DAF4 primers for 25 isolates of Acinetobacter spp. obtained from 16 different hospitals situated in 12 countries. The overall robustness of the algorithms and the reproducibility of the cluster analysis results generated by two commercially available computer programs (GelCompar and DENDRON) for analysing DNA fingerprinting gels were tested by examining the same set of fingerprinting data independently in two laboratories with the different software packages. Both programs were efficient at recognising and grouping strains with closely similar RAPD fingerprints, i.e., strains which might be expected to have a close epidemiological or evolutionary relationship. However, the relationships suggested for less closely related strains showed considerable variation in terms of the overall similarity or percentage correlation values suggested by the programs. It was concluded that both programs were useful tools for indicating close genotypic relationships between individual strains, but that epidemiological conclusions based on the similarity or correlation values (or the dendrograms derived from them) obtained for less closely related strains should be treated with considerable caution.

Acinetobacter↗
PubMed2005

Application of sliding-window discretization and minimization of stochastic complexity for the analysis of fAFLP genotyping fingerprint patterns of Vibrionaceae.

Minimization of stochastic complexity (SC) was used as a method for classification of genotypic fingerprints. The method was applied to fluorescent amplified fragment length polymorphism (fAFLP) fingerprint patterns of 507 Vibrionaceae representatives. As the current BinClass implementation of the optimization algorithm for classification only works on binary vectors, the original fingerprints were discretized in a preliminary step using the sliding-window band-matching method, in order to maximally preserve the information content of the original band patterns. The novel classification generated using the BinClass software package was subjected to an in-depth comparison with a hierarchical classification of the same dataset, in order to acknowledge the applicability of the new classification method as a more objective algorithm for the classification of genotyping fingerprint patterns. Recent DNA-DNA hybridization and 16S rRNA gene sequence experiments proved that the classification based on SC-minimization forms separate clusters that contain the fAFLP patterns for all representatives of the species Enterovibrio norvegicus, Vibrio fortis, Vibrio diazotrophicus or Vibrio campbellii, while previous hierarchical cluster analysis had suggested more heterogeneity within the fAFLP patterns by splitting the representatives of the above-mentioned species into multiple distant clusters. As a result, the new classification methodology has highlighted some previously unseen relationships within the biodiversity of the family Vibrionaceae.

Algorithms↗
PubMed1995

DNA fingerprinting of crude bacterial lysates using degenerate RAPD primers.

Methods for identifying isolates of various pathogenic bacteria by DNA fingerprinting with random primers (RAPD) have been described recently. In these methods many primers are screened and the primers that generate the most informative DNA pattern are selected. A new strategy that simplifies the primer selection process for RAPD fingerprinting has been developed in our laboratory. In this approach, one or more degenerate nucleotides is introduced into the core RAPD primer sequence at various nucleotide positions. Results show that a single degenerate nucleotide in the primer sequence can significantly change the DNA profile obtained for the same template. The more removed the degenerate nucleotide is from the 3' end of the primer, the less dramatic is its effect on banding pattern. This method utilizing degenerate RAPD (D-RAPD) primers was tested on clinical isolates of Legionella pneumoniae, and results were confirmed with nondegenerate RAPD primers. Results obtained with D-RAPD primers were in total agreement with those obtained with nondegenerate RAPD primers. We propose that the use of a core RAPD primer sequence with one or more degenerate nucleotide(s) at various positions can expedite the generation of unique DNA fingerprints individual organisms. A general method for selecting the most useful fingerprinting RAPD primers is discussed.

Base Sequence↗
PubMed1991

DNA fingerprinting in cattle using oligonucleotide probes.

Oligonucleotide probes specific for simple tandem repeat sequences produce individual specific DNA fingerprints in man and all animal species tested so far. Here 11 different synthetic probes were hybridized to bovine genomic DNAs which had been digested with the restriction endonucleases HinfI, AluI and HaeIII. Two of these probes gave DNA fingerprint patterns which were analysed for three German breeds. Different parameters were calculated, such as the average number of bands per individual or the probability of finding identical fingerprints in two unrelated individuals. The number of polymorphic bands varies from 11 to 23 in the different breeds and the probability of finding the same banding pattern in two unrelated individuals ranges from 1.5 x 10(-7) to 2.4 x 10(-7). Hence this DNA fingerprinting procedure allows precise identification of individuals. It is also a useful additional method for paternity testing in cattle.

Animals↗
PubMed1993

Assessment of DNA fingerprinting for determining genetic diversity in sheep populations.

Genomic DNA, prepared from 12 animals from four sheep flocks, was digested with either HaeIII or HinfI and probed with three DNA fingerprinting probes. Mean DNA fingerprint band sharing and band frequency calculated for each flock were used to estimate genetic diversity. Each of the DNA fingerprinting systems showed the same trend in diversity within the sampled flocks, and greater diversity between the flocks than within the flocks. DNA fingerprinting therefore provides a useful measure of genetic diversity in sheep.

Analysis of Variance↗
PubMed1993

A novel molecular fingerprint probe based on the endogenous avian retroviral element (EAV) of chickens.

We have developed a novel molecular probe that is useful for DNA fingerprint analysis in chickens. The probe is based on the middle-repetitive, chicken endogenous retroviral (EAV) element. It consists of 1503 bp of the 3' portion of the EAV element, extending from the down-stream end of the envelope gene to the beginning of the downstream long terminal repeat (LTR). Unlike other probes that are currently in use for fingerprint analysis with chicken DNA, the EAV-based probe works well at normal levels of stringency, and with standard hybridization buffers. Digestion of chicken genomic DNA with a variety of restriction enzymes routinely yields up to 30 resolvable bands per bird in the 500 bp to 20 kbp range. In order to test the efficacy of the EAV-based fingerprint probe, we have used it to estimate the degree of inbreeding in the inbred WG strain of White Leghorns. We find that the estimates derived with the EAV probe are very similar to those reported previously for the WG strain. These results suggest that molecular probes based on endogenous retroviruses and other middle-repetitive DNA elements should be useful for fingerprint analysis in chickens, and in vertebrates in general.

Animals↗
PubMed1996

Genomic DNA fingerprinting of clinical isolates of Helicobacter pylori using short oligonucleotide probes containing repetitive sequences.

The ability of oligonucleotide probes containing short repetitive sequence motifs to differentiate between isolates of Helicobacter pylori was investigated. Genomic DNA preparations from H. pylori were digested with the restriction enzyme HindIII, electrophoresed in agarose gels and transferred to nylon filters. Five separate oligonucleotide probes were tested for hybridization sequentially to fingerprint the digested DNA from a panel of 29 clinical isolates and one type strain of H. pylori, and their relative discriminatory abilities were assessed. Four probes, (GACA)4, (GT)8, (GTG)5 and (GGAT)4, were each shown to yield highly informative hybridization band profiles allowing differentiation of H. pylori isolates. The DNA fingerprints of individual isolates obtained with each probe were distinct and reproducible. Direct comparison with ribotyping revealed that oligonucleotide fingerprinting had far superior discriminatory power. Computer-assisted similarity analysis of (GGAT)4-generated hybridization profiles of pairwise combinations of H. pylori isolates revealed that there was no correlation between ribotype and oligonucleotide fingerprint patterns. The results of this study demonstrate that oligonucleotide probes containing microsatellite sequences provide a new and powerful tool for isolate discrimination of H. pylori.

DNA Fingerprinting↗
PubMed2005

Ochratoxin A producing Penicillium verrucosum isolates from cereals reveal large AFLP fingerprinting variability.

AIMS: To examine if molecular amplified fragment length polymorphism (AFLP) fingerprinting of the only ochratoxin A-producing species in European cereals, Penicillium verrucosum, can be used as a method in hazard analysis using critical control points (HACCP). METHODS AND RESULTS: A total of 321 isolates of P. verrucosum were isolated from ochratoxin A-contaminated cereals from Denmark (oats), UK (wheat and barley) and Sweden (wheat). Of these, 236 produced ochratoxin A as determined by thin layer chromatography; 185 ochratoxin A-producing isolates were selected for AFLP fingerprinting. A total of 138 isolates had unique AFLP patterns, whereas 52 isolates could be allocated to small groups containing from two to four isolates with similar AFLP patterns. A total of 155 clones were found among the 185 P. verrucosum isolates, thus 84% of the isolates may represent different genets of P. verrucosum. As the few isolates that were grouped often came from the same farm, and those groups that contained AFLP-identical isolates from different countries were morphotypically different. On single farms up to 35 clones were found. The few groups of ramets from the same genet indicated that a HACCP approach based on clones may require a very large number of AFLP analysis to work in practice, we recommend basing the HACCP approach on the actual species P. verrucosum. A more detailed characterization should rather be based on the profile of species present at different control points, or analysis of the mycotoxins ochratoxin A and citrinin in the isolates. Examination of 86 isolates with HPLC and diode array detection of P. verrucosum showed that 66% produced ochratoxin A, 87% produced citrinin, 92% produced verrucin and 100% produced verrucolone. CONCLUSIONS: Among 184 ochratoxin A-producing Penicillium verrucosum, 155 clonal lineages were indicated by AFLP fingerprinting, indicating a high genetical diversity, yet the species P. verrucosum is phenotypically distinct and valid. SIGNIFICANCE AND IMPACT OF THE STUDY: AFLP fingerprinting of Penicillium verrucosum indicates that genetic recombination takes place in this fungus.

Avena↗
PubMed1994

Differentiation of aphid clones by arbitrarily primed polymerase chain reaction (AP-PCR) DNA fingerprinting.

Differentiation of aphid clones was attempted using AP-PCR which is a simple and rapid method to obtain DNA fingerprints of complex genomes. To establish optimal reaction conditions and examine reproducibility of the method, a laboratory-maintained clone of the pea aphid, Acyrthosiphon pisum, was used as test material. Under the reaction conditions employed, identical fingerprint patterns were obtained throughout a wide range of template DNA amount, from 5 to 800 ng, and irrespective of aphid instar. No changes in the patterns were seen throughout five parthenogenetic generations. When this method was applied to a wild population of the gall-forming aphid, Ceratovacuna nekoashi, five groups of insects originating from different galls formed on the same twig were successfully differentiated from one another by means of polymorphic fingerprint bands. In contrast, the fingerprints of the insects derived from the subgalls of the same gall were identical. These results indicated that in C. nekoashi: (i) members of a gall constitute a clonal population; (ii) a gall is founded by a single fundatrix; and (iii) intergall migration is absent or at least not frequent.

Animals↗
PubMed1996

Differentiation of Pasteurella multocida subspecies multocida isolates from the respiratory system of pigs by using polymerase chain reaction fingerprinting technique.

PCR fingerprinting technique was applied to subtype 44 Pasteurella multocida subspecies multocida (P.m.sp.m.) isolates from the respiratory system of pigs. Two single primers were tested for their abilities to generate individual fingerprints by using PCR. Primer 1 (core sequence of the M13 phage) grouped the 44 P.m.sp.m. strains into five distinct fingerprinting profiles, while primer 2 ((GACA)4) grouped them into seven profiles. The results suggest that PCR fingerprinting is an efficient technique to detect DNA polymorphism in the species P.m.sp.m. This technique could be used to differentiate P.m.sp.m. strains of the same capsular serotype.

Animals↗
PubMed2005

Retrieval of nearly complete 16S rRNA gene sequences from environmental DNA following 16S rRNA-based community fingerprinting.

16S rRNA-based fingerprinting techniques allow rapid analyses of overall bacterial community structure but suffer from a lack of phylogenetic information hitherto retrievable from the short 16S rRNA gene sequences obtained from excised bands. An approach is presented that allows nearly complete 16S rRNA gene sequences to be retrieved for abundant components of the bacterial community as obtained by the community fingerprint, i.e. those reflected by major fingerprint bands. This was achieved by designing a pair of highly specific primers derived from the sequence of an excised band. Combined with universal 16S rRNA primers, these specific primers were applied directly to environmental DNA serving as template. This procedure allowed the generation of a nearly complete 16S rRNA gene sequence of the target taxon by specific polymerase chain reaction (PCR) followed by cycle sequencing down to a relative abundance of at least 1.5% of the environmental DNA. The procedure was exemplified for an epsilonproteobacterium related to Thiomicrospira denitrificans occurring in the central Baltic Sea. This approach is based only on PCR without any cloning step involved. It allows focussing on specific target taxa and is thus rather efficient. This approach should be applicable in general to 16S rRNA or 16S rRNA gene-based fingerprinting techniques and their respective environmental DNA.

DNA Fingerprinting↗