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PubMed2003

Normalizing the background and removing the trend in one-dimensional DNA fingerprint images.

Maximizing an individual's genetic information from its DNA fingerprint image depends on the number of bands distinguished from the background. To approach this goal, the background should be normalized while the information is preserved. Morphological operators have been used by some authors to normalize the background for two-dimensional gel images. Methods such as mean, median and "maxpolygon" are presented in this work to normalize the background in DNA fingerprint images. Mean and median methods will lead to some deformations. Visual evaluation of the results show that the original shape of the column signals are better preserved by the maxpolygon.

DNA Fingerprinting↗
PubMed1999

An algorithm for the assembly of robust physical maps based on a combination of multi-level hybridization data and fingerprinting data.

We have developed an algorithm which combines data obtained from restriction digestion experiments and hybridization experiments to construct robust physical maps of whole chromosomes. The algorithm has been incorporated into a program which accepts hybridization data consisting of an unordered hybridization matrix and fingerprinting data containing band coordinates for each clone. The combined data is used to produce a non-redundant, ordered matrix which can be further reduced to represent a minimum tile coverage of the chromosome. In addition, the method also takes into account multi-level hybridization events which allows for an improved treatment of the hybridization data. The program is evaluated against several other contig building programs using simulated and real data sets. Finally, it is applied to construct a physical map of the 4.1 Mb genome of Ochrobactrum anthropi based on 1387 clones and 70 probes, as well as 624 fingerprints.

Algorithms↗
PubMed2001

Design and evaluation of PCR primers to amplify bacterial 16S ribosomal DNA fragments used for community fingerprinting.

Denaturing gradient gel electrophoresis of PCR-amplified 16S ribosomal DNA (rDNA) fragments has frequently been applied to the fingerprinting of natural bacterial populations (PCR/DGGE). In this study, sequences of bacterial universal primers frequently used in PCR/DGGE were compared with 16S rDNA sequences that represent recently proposed divisions in the domain Bacteria. We found mismatches in 16S rDNA sequences from some groups of bacteria. Inosine residues were then introduced into the bacterial universal primers to reduce amplification biases caused by these mismatches. Using the improved primers, phylotypes affiliated with Verrucomicrobia and candidate division OP11, were detected in DGGE fingerprints of groundwater populations, which have not been detected by PCR/DGGE with conventional universal primers.

Bacteria↗
PubMed2001

AFLP fingerprinting as a tool to study the genetic diversity of Rhizobium galegae isolated from Galega orientalis and Galega officinalis.

AFLP fingerprints of Rhizobium galegae strains that infect Galega orientalis and Galega officinalis obtained from different geographical sources, and of taxonomically diverse rhizobia representing the recognized species, were generated. Comparisons of the fingerprints from fluorescent labeled AFLP products using capillary electrophoresis on ABI prism 310, slab gel electrophoresis on ABI prism 377 genetic analyzers and silver staining were in good agreement. All methods delineated the G. orientalis strains from G. officinalis strains, the G. orientalis strains formed a tight cluster whereas the G. officinalis strains seem to show a greater level of genetic diversity. Comparison of fluorescent AFLP with other detection methods revealed that fluorescent labeling is more sensitive and practical, in addition, the deleterious effect of radioactivity associated with 32P-labeling, the delicate process of blotting polyacrylamide gels or the tedious procedure of silver staining can be avoided. The automated system facilitated a large number of runs at a time and the subsequent analysis of the data by generating exportable raw data. The congruency of the experiments was analyzed using the Bionumerics software.

DNA Fingerprinting↗
PubMed1998

Intra-species differentiation of Trypanosoma evansi by DNA fingerprinting with arbitrary primered polymerase chain reaction.

A method was developed to obtain reproducible DNA fingerprints from isolates of Trypanosoma evansi by PCR-based amplification using arbitrary primers (AP-PCR). Only one out of 10 randomly designed 12-mer primers generated DNA fingerprint profiles that revealed intra-species differences in T. evansi. The technique was applied in association with parasitological and serological examinations to investigate animal-to-animal transmission during an outbreak of surra in Thailand. The AP-PCR method has the advantage of being simple, fast and sensitive to diagnosis and characterization of the parasites since it does not require prior DNA sequence information. The technique should prove useful for the proper understanding of epidemiology and for designing rational control programs for trypanosomosis.

Animals↗
PubMed2003

Optimisation of AP-PCR fingerprinting discriminatory power for clinical isolates of Pseudomonas aeruginosa.

Recently methods based on analysis of arbitrarily amplified target sites of microorganism genomes have been extensively applied in microbiological studies. The range of their applications is limited by problems with discrimination and reproducibility resulting from lack of standardised and reliable methods of optimisation. By orthogonal-array optimisation most advantageous and optimal parameters for highly discriminatory primers (CagA2+CMVin2) were selected and efficient AP-PCR (arbitrarily primed-polymerase chain reaction) fingerprinting conditions for Pseudomonas aeruginosa isolates were set up. Stable and multiplex amplicon profiles obtained in this study revealed high level of intraspecies DNA polymorphism among 20 analysed clinical strains of P. aeruginosa proving optimised AP-PCR fingerprinting to be useful in epidemiological typing of the species.

DNA Fingerprinting↗
PubMed1999

Analyses of lipopolysaccharides, outer membrane proteins and DNA fingerprints reveal intraspecies diversity in Moraxella bovis isolated in Argentina.

Intra-specific diversity within Moraxella bovis was investigated analysing DNA fingerprints, outer membrane proteins (OMP) and lipopolysaccharides (LPS) profiles. Three collection strains and 57 isolates of M. bovis, collected during 3 years from cattle with infectious bovine keratoconjunctivitis (IBK) symptoms, from diverse geographical locations of Argentina, were examined. The LPS and OMP profiles were studied through SDS-PAGE analysis and genotype was determined by PCR-DNA fingerprinting. Genotyping identified five DNA types while analysis of LPS and OMP profiles identified three rough LPS types and three OMP types among the 60 isolates of M. bovis including the three collection strains. None of the three methods employed to assess diversity was discriminating when used alone because the degree of heterogeneity in each group of surface structures was limited, but when data of each typing method were combined, 15 distinct subgroups were determined. This subgrouping was clearly able to differentiate isolates of the same genotype. These typing methods appear to be useful to assess different aspects of the disease such as the diversity within a population of M. bovis associated to epidemic conditions, track the causal agent in an outbreak of the disease, monitoring vaccination programs and studies on virulence.

Animals↗
PubMed1999

Molecular fingerprinting confirms extensive cow-to-cow intra-herd transmission of a single Mycobacterium bovis strain.

In this study we have characterized M. bovis isolates from a herd of cattle in Uvalde, Texas in which 52 of the 193 animals selected at random in 1994 from a herd of 331 were caudal fold skin-test positive. Thirty-two of 52 skin-test positive cattle had gross lesions at slaughter, and isolations of M. bovis were made from 29 animals. The herd was comprised of Red Devon cattle purchased between 1978 and 1980 (n = 26) and breeding bulls (n = 3) introduced at later times, and all were tuberculosis test negative at the time of purchase. Other animals were natural additions (offspring) of these cattle. One additional animal, a Holstein present on the ranch at the time of purchase in 1976, was retained to nurse orphaned and weak calves. Using several molecular fingerprinting techniques we have verified a clonal relationship among the M. bovis isolates consistent with infection originating with a single strain. The molecular fingerprint patterns demonstrate the stability of the profiles despite persistence and spread of the organism within the herd for two decades and confirms their use in epidemiological tracing.

Animals↗
PubMed2000

AFLP fingerprint analysis of Bradyrhizobium strains isolated from Faidherbia albida and Aeschynomene species.

The diversity of Bradyrhizobium isolates from Faidherbia albida and Aeschynomenee species was assessed using AFLP analysis, a high-resolution genomic fingerprinting technique. Reference strains from Bradyrhizobium japonicum, Bradyrhizobium elkanii and Bradyrhizobium liaoningense were included for comparison. At a similarity level of 50%, a total of 34 different groups were obtained by cluster analysis of the genomic fingerprints. Four of these clusters correspond to the three reference species, demonstrating the large diversity of the isolates studied. Comparison with other data demonstrates that AFLP has a higher resolution than restriction analysis of 16S rRNA genes, SDS-PAGE analysis of proteins and phenotypic analysis. Results of the latter two methods showed little correspondence with the genotypic data.

Acacia↗
PubMed2000

PCR fingerprinting analysis for differentiation of Streptococcus pneumoniae reinfection versus relapse.

Polymerase chain reaction fingerprint profiles of isolates obtained during an episode of pneumococcal pneumonia with bacteremia differed significantly from profiles of isolates obtained from the same patient during a subsequent episode of pneumococcal meningitis with bacteremia. Polymerase chain reaction fingerprinting provides a means of differentiating new infection from relapse, and may be a simple molecular tool for comparison of Streptococcus pneumoniae isolates.

Bacteremia↗
PubMed1994

Use of small fragment restriction endonuclease analysis (SF-REA) for epidemiological fingerprinting of Klebsiella oxytoca.

A group of infections caused by Klebsiella oxytoca was observed among preterm neonates in a neonatal intensive care unit (NICU) of a pediatric hospital in Osnabrück, Germany. The presence of unique antimicrobial susceptibility patterns among the bacterial isolates prompted an investigation to determine whether a limited spread of one single strain existed. All 4 K. oxytoca isolates from the NICU and, in addition, 50 epidemiologically non-related strains were fingerprinted by small fragment restriction endonuclease analysis (SF-REA). From an analysis of silver-strained EcoR I-generated restriction fragment patterns of whole-cell DNA in polyacrylamide gels, it was evident that the 4 NICU isolates were closely related, whereas all epidemiologically unrelated strains exhibited different patterns. From the results of this study, DNA fingerprinting by SF-REA is proposed as a useful tool for investigating the epidemiological relatedness of K. oxytoca strains of clinical and environmental origin.

Bacterial Typing Techniques↗
PubMed1999

The properties of human DNA fingerprints produced by polymeric monocore probes (PMC probes).

The properties of human DNA fingerprints detected by multilocus polymeric monocore probes (PMC probes) have been investigated. The PMC probes were produced by the polymerase chain reaction with two partly complementary oligonucleotides homologous to various minisatellite or microsatellite core sequences (Ijdo J, Wells RA, Baldini A, Reeders ST. Nucleic Acids Res 1991;19:4780). It has been shown that these probes possess increased sensitivity, they detect considerably more hypervariable fragments in genomic DNA thus exhibiting advantages over the corresponding oligonucleotides and natural polycore minisatellite probes. Variation in the DNA fingerprints of different individuals produced by these probes indicates that the probability of accidental identity is very low (< 10(-12)). According to the data of cross-hybridization with PMC probes of various specificity, several distinct families can be distinguished among G-rich hypervariable sequences of the human genome.

Bacteriophage M13↗
PubMed2002

Whole genome fingerprinting and genotyping of multiple drug resistant (MDR) isolates of Pseudomonas aeruginosa from endophthalmitis patients in India.

Genome sequence-based fluorescent amplified-fragment length polymorphism (FAFLP) analysis was investigated for fingerprinting and subtyping 42 multiple drug resistant (MDR) isolates of Pseudomonas aeruginosa from post-surgical endophthalmitis. The FAFLP profiles derived from EcoRI/MseI restricted fragments differentiated clinical isolates and were found to be extremely reproducible. Seventeen different amplification patterns (amplitypes) were observed for all the 42 isolates from 11 different patients. Also, we were able to genotype the isolates based on the differential amplification of a total of 31 FAFLP markers representing genomic fragments from the P. aeruginosa chromosome. This data appears to provide clues to the genetic diversity of endopthalmitis associated strains and could be converted into digitised fingerprints suitable for electronic manipulations and archiving.

DNA Fingerprinting↗
PubMed1995

Random amplified polymorphic DNA fingerprints of the eight taxa of Trichinella and their comparison with allozyme analysis.

Eight taxa have recently been proposed as being encompassed by the genus Trichinella on the basis of allozyme and biological data. In this paper we show that an analogous 8 taxon structure for this genus results from the random amplified polymorphic DNAs (RAPDs). Five 10-mer or 20-mer primers were used under different polymerase chain reaction (PCR) conditions to produce multiband RAPD fingerprints from muscle larvae of 40 isolates of Trichinella spp. The resulting RAPD data were analysed following the numerical taxonomic approach, and the resulting classification was compared to that derived from allozyme data. The agreement found between allozymes and RAPDs, while supporting the polyspecific structure of the genus Trichinella, confirms the potential of RAPDs as a tool for the detection of cryptic species. The selected primers were tested on individual muscle larvae in an attempt to standardize a RAPD assay for the routine identification of the 8 taxa of Trichinella. Only 1 of the 5 primers yielded reproducible fingerprints from the single larvae. Using this primer, the 5 species and the 3 other taxa of the genus Trichinella can be identified in a single assay without the need for massive in vivo parasite production.

Animals↗
PubMed1996

Usefulness of the DNA-fingerprinting pattern and the multilocus enzyme electrophoresis profile in the assessment of outbreaks of meningococcal disease.

The objective of the study was to assess whether genotypic characterization by means of DNA-fingerprinting pattern (DFP) and multilocus enzyme electrophoresis (MEE) profile as compared to phenotypic characterization would improve the differentiation of Neisseria meningitidis strains associated with outbreaks from strains associated with sporadic cases of meningococcal disease. In addition, the differentiation of serogroup C carrier strains from those associated with an outbreak of serogroup C meningococcal disease was investigated. A total of 118 N. meningitidis strains were available for the study: 59 from patients involved in outbreaks of meningococcal disease (2 serogroup B and 2 serogroup C), 37 patients considered to be sporadic cases and 22 serogroup C carrier strains. Among the 59 strains from patients involved in outbreaks the 4 strains isolated from the patient registered as the first in each outbreak were designated the index strains. Among the remaining 55 outbreak strains 52 were either DFP-identical or DFP-indistinguishable when compared with the one relevant out of the 4 index strains. This was only the case for 17 of the 37 strains isolated from sporadic cases caused by the same serogroup of meningococci during the outbreak periods, and 5 of the 22 meningococcal strains isolated from healthy carriers. Among the 56 (52 + 4) DFP-identical or DFP-indistinguishable outbreak strains 5 different electrophoretic types were identified by MEE. Among 59 assumed outbreak strains a total of 4 were identified as genotypically distinct. Among the 37 mainly DFP-indistinguishable or DFP-different strains from sporadic cases 17 different ETs were identified, and among the 22 mainly DFP-different carrier strains 13 different ETs were identified. Two strains among those selected from sporadic cases were identical to the outbreak strain. None of the local serogroup C carrier strains isolated during the outbreak of serogroup C disease were identical to the outbreak strain. Both DNA-fingerprinting and MEE improved the differentiation of meningococci when compared with phenotypic characterization. The results indicate that tracing a virulent strain within a open group of contacts is irrelevant.

Bacterial Typing Techniques↗
PubMed1998

M genotyping and DNA fingerprinting of Streptococcus pyogenes isolates from an area of central Italy.

M protein gene typing was used to analyse Streptococcus pyogenes clinical isolates collected between 1983 and 1995 in an area of central Italy from patients presenting different types of infections; the same isolates were also characterized by means of DNA fingerprinting. M type 1 was the most common (50% of study strains), followed by M types 4, 12 and 6. The proportion of M type 12 decreased with time, whereas M type 1 increased, in agreement with data obtained in many different areas. Most invasive strains belonged to types M1 (30%) and M12 (30%); on the other hand, the M1 type did frequently occur also among non-invasive isolates. DNA fingerprinting showed a correlation between M types and DNA patterns. This report provides epidemiological information from a geographic area not sampled recently, and further shows the usefulness of the M genotyping technique, which offers potential advantages over conventional serological typing methods.

DNA Fingerprinting↗
PubMed1999

Application of ribotyping and IS200 fingerprinting to distinguish the five Salmonella serotype O6,7:c:1,5 groups: Choleraesuis sensu stricto, Choleraesuis var. Kunzendorf, Choleraesuis var. Decatur, Paratyphi C, and Typhisuis.

Sixty-seven strains of the five described Salmonella serotypes having antigens 6,7:c:1,5, that is S. enterica serotype Choleraesuis sensu stricto, Choleraesuis var. Kunzendorf, Choleraesuis var. Decatur, Paratyphi C, and Typhisuis, were examined for 16S rrn profile ribotype, presence of IS200 and phenotypic characters, including rate of change of flagellar-antigen phase and nutritional character. Choleraesuis sensu stricto and its Kunzendorf variant had related but distinct ribotypes. Therefore, ribotyping appears to be a suitable method for differentiating Choleraesuis non-Kunzendorf from Choleraesuis var. Kunzendorf. Some strains of Paratyphi C had 16S profiles that resembled that of Choleraesuis non-Kunzendorf, while others resembled that of Choleraesuis var. Kunzendorf. The Typhisuis profiles were like those of Choleraesuis non-Kunzendorf, while the Choleraesuis var. Decatur profiles were unlike those of any of the other four groups. Furthermore, IS200 fingerprinting discriminated between Choleraesuis var. Decatur and the other strains with antigenic formula O6,7:c:1,5, and comparison of IS200 patterns showed a high degree of genetic divergence within Choleraesuis var. Decatur. Our findings show that ribotyping and IS200 fingerprinting, combined with classical microbiological methods, distinguish the groups Choleraesuis non-Kunzendorf, Choleraesuis var. Kunzendorf, Choleraesuis var. Decatur, Paratyphi C and Typhisuis.

Animals↗
PubMed2002

Short-term changes in bacterial community fingerprints and potential activities in an alfisol supplemented with solid waste leachates.

We investigated the effect on soil functioning of adding leachates from municipal solid waste incinerator (MSWI) ashes to laboratory columns (bare soil) and to field experimental plots (bare soil or grass cover). Leachate of MSWI-solidified air pollution control residues (SAPCr) contained more salts but less heavy metals than did MSWI-bottom ash (BA) leachate. Leachate-supplemented soils (BA soil, SAPCr soil) and control (water added) soil (W) were analyzed after 30 days. Potential denitrifying activity (PDA) and potential N2 fixation (acetylene reduction assay, ARA) were measured in controlled conditions. PDA was significantly lower in SAPCr soil than in W soil, both in the laboratory (-45%) and in bare soil in the field (-31%). ARA values were lower in bare SAPCr soil (-54%) and in bare BA (-25%) soil. Both activities remained unaffected by leachate supplementation in soil under permanent grass cover. Automated ribosomal intergenic spacer analysis (A-RISA) fingerprints and RFLP of nifH gene pools were used to assess changes in the structure of bacterial community. Multivariate analysis of these fingerprints revealed that SAPCr leachate had a stronger effect than BA leachate on the total and N2-fixing bacterial communities. Similar results were obtained for laboratory and bare soil field plots, but leachates did not affect nifH gene pools from soil under permanent grass cover. The stronger impact of SAPCr leachate on both structure of bacterial communities and activities supports the conclusion that observed effects would result from the abundance of salts rather than from heavy metal toxicity.

Bacteria↗