DNA Fingerprint Patterns Research Articles

DNA Fingerprints From Mycobacterium tuberculosis Isolates of Patients Confined for Therapy Noncompliance Show Frequent Clustering

To test the hypothesis that individuals chronically noncompliant with antituberculous chemotherapy are vectors for ongoing transmission of the disease in the community. Cohort study. A large public hospital with a tuberculosis detention unit for patients with repeated and prolonged nonadherence to therapy. Mycobacterium tuberculosis isolates from patients confined on the detention unit were obtained from the hospital's mycobacteriology laboratory. None. A standardized IS6110-based Southern blot hybridization protocol was used to genotype M tuberculosis isolates recovered from patients confined on the detention unit at the hospital. Each DNA fingerprint pattern was compared with the IS6110-fingerprint database at the Public Health Research Institute Tuberculosis Center, which has archived fingerprint patterns from over 2,500 M tuberculosis isolates collected from New York City patients in the past 5 years. Eighty percent of available isolates from detained patients belonged to an identifiable DNA fingerprint cluster, suggesting an epidemiologic link between the detainees and other New York City tuberculosis patients. Chronic noncompliance with therapy is associated with ongoing spread of tuberculosis in the community. Aggressive measures, including detention, for the small number of recalcitrant, noncompliant patients may interrupt a chain of transmission and contribute to a decline in the spread of tuberculosis in urban areas.

PCR primed with minisatellite core sequences yields DNA fingerprinting probes in wheat

Four minisatellite core sequences were used as primers in a polymerase chain reaction (PCR) technique, known as the directed amplification of minisatellite-region DNA (DAMD), to detect polymorphisms in three pairs of hexaploid/tetraploid wheat cultivars. In each pair, the tetraploid cultivar (genomic formula AABB) was extracted from its corresponding hexaploid (genomic formula AABBDD) parent. Reproducible profiles of the amplified products revealed characteristic bands that were present only in the hexaploid wheats but not in their extracted tetraploids. Some polymorphisms were observed among the hexaploid cultivars. Twenty-three DAMD-PCR amplified fragments were isolated and screened as molecular probes on the genomic DNA of wild wheat species, hexaploid wheat and triticale cultivars. Subsequently, 8 of the fragments were cloned and sequenced. The DAMD-PCR clones revealed various degrees of polymorphism among different wild and cultivated wheats. Two clones yielded individual-specific DNA fingerprinting patterns which could be used for species differentiation and cultivar identification. The results demonstrated the use of DAMD-PCR as a tool for the isolation of informative molecular probes for DNA fingerprinting in wheat cultivars and species.

PCR-based DNA fingerprinting of Staphylococcus haemolyticus to investigate nosocomial infections.

OBJECTIVE: To apply PCR-based DNA fingerprinting in a clinical microbiology laboratory to investigate nosocomial infections with Staphylococcus haemolyticus. METHODS: DNA fingerprints were generated by PCR on 99 S. haemolyticus isolates using different primer combinations based on ERIC, REP or arbitrarily chosen simple repeat sequences. RESULTS: Primer combinations REP1+(GTC)6 and ERIC1+ERIC2 had sufficient discrimatory power and were chosen to analyze the clinical isolates. DNA fingerprint patterns from strains isolated from the patients nursed in the same hospital ward in the period 1991--94 were approximately 90% similar to each other. One staff member, sampled in 1991, carried a strain with a similar fingerprint. CONCLUSIONS: PCR based DNA fingerprinting is a suitable method to perform in a clinical laboratory. An S. haemolyticus strain appeared to be endemic in the hospital ward and had most probably been transmitted from patient to patient. S. haemolyticus may carry glycopeptide resistance and needs attention as a causative agent of nosocomial infections.

Clonal Atlantic salmon × brown trout hybrids produced by gynogenesis

Clonal full‐sib progeny groups of Atlantic salmon Salmo salar× brown trout Salmo trutta hybrids were produced by gynogenesis. Eggs obtained from two 3‐year‐old Atlantic salmon (female) × brown trout (male) F1 hybrids were activated with UV‐irradiated rainbow trout Oncorhynchus mykiss sperm. Fecundity, percentage egg activation and percentage survival to completion of yolk‐sac absorption were similar for the two females, and averaged 800 eggs kg−1, 90 and 65%, respectively. Flow cytometric and protein electrophoretic analyses confirmed the progeny to be diploid hybrids. Isogenicity within progeny groups and to the maternal parent was indicated by identical DNA fingerprint patterns detected with multilocus oligonucleotide probes–GATA(5) and ACTG(n). Isogenicity was also observed in the gynogenetic progeny of a third female spawned the following year. It appeared that a large portion of the oocytes in females of this hybrid underwent a premeiotic chromosome doubling, or possibly a complete suppression of meiosis. The result was ovulation of diploid eggs, each possessing a full set of both Atlantic salmon and brown trout chromosomes identical to those in the maternal somatic cells. Lines of clonal hybrids could therefore be perpetuated by gynogenesis and would have potential both as experimental animals and in commercial aquaculture.

DNA analysis of Cylindrocladium floridanum isolates from selected forest nurseries

Isolates of Cylindrocladium floridanum obtained from diseased roots of white and black spruce seedlings and surrounding soil in bareroot nurseries in Ontario, Minnesota and Wisconsin area were compared by mitochondrial DNA (mtDNA) and ribosomal DNA (rDNA) restriction fragment length polymorphisms (RFLPs), human minisatellite DNA fingerprints and polymerase chain reaction. Nucleotide sequences of rDNA ITS regions of selected isolates were also performed. Based on the nuclear DNA fingerprinting patterns, these isolates could be divided into three groups, namely S1, S2 and S3. Isolates of S1 and S2 showed the same mtDNA RFLPs which were slightly different from that of S3. Isolates representing each of the three groups showed an identical nucleotide sequence of rDNA ITS regions. These data suggest that all the isolates tested belong to the same species which may be divided into three biotypes or populations.

Identification of Streptococcus Species by Randomly Amplified Polymorphic Deoxyribonucleic Acid Fingerprinting

Identification of Streptococcus species isolated from bovine milk by randomly amplified polymorphic DNA fingerprinting was evaluated. Bacterial DNA templates were prepared using a standardized method for randomly amplified polymorphic DNA amplification and previously evaluated arbitrary primers. Amplified DNA fragments were visualized by agarose gel electrophoresis and were analyzed by densitometry. Unidentified streptococci (n = 163) that were isolated from mammary secretions of dairy cows were evaluated. The DNA fingerprint patterns of unidentified bacteria were compared using a computerized data-base that contained DNA fingerprint patterns of test strains. Comparison with the API 20 Strep identification system (bioMérieux Vitek, Inc., Hazelwood, MO) and conventional biochemical tests showed that about 91% of isolates (148 of 163) were identified correctly by DNA fingerprinting. The sensitivity of the DNA fingerprinting technique was 90%, and the specificity was 92%. However, the DNA fingerprinting technique only identified 4 of the 11 species included in the study. Three of the 4 species, Streptococcus uberis, Streptococcus agalactiae, and Streptococcus dysgalactiae, represent the streptococci isolated most frequently from cows with mastitis. The other Streptococcus and Enterococcus species that were not identified by the DNA fingerprinting system are less frequently isolated as causative agents of mastitis. Expanding the DNA fingerprint database would likely increase the sensitivity and specificity of this technique.

Genetic relationships within avocado (Persea americana Mill) cultivars and between Persea species

The origin of avocado (Persea americana Mill) cultivars, as well as the genetic relationships between Persea species, are not well defined and are based mainly on morphological parameters. Minisatellite DNA markers were used to analyze 24 P. americana cultivars in an attempt to define their racial allocation. DNA mixes representing the three races were evaluated and used for analysis. The allocation of 19 of the above cultivars was substantiated by the DNA markers, while new suggestions were offered regarding the remaining five. Eight cultivars, of unknown racial origin, were also examined, and a phylogenetic tree suggesting their origin is offered. Selfing progeny of five cultivars were analyzed for six morphological traits which differentiate the three races, and were compared to their parents in order to assess their origin. Eleven Persea species were analyzed, using DNA fingerprint patterns and SSR (simple sequence repeat) alleles, in order to identify the genetic relationships among the Persea species, and between them and the three P. americana races. The phylogenetic tree obtained is presented. The high value of variation between the avocado cultivars and Persea species observed in this work, suggests that the validity of race and species definition within Persea be treated with caution.

DNA fingerprinting in Indian major carps and tilapia by Bkm 2(8) and M13 probes

DNA fingerprints were obtained in three species of commercially important freshwater fishes, Labeo rohita (Hamilton). Catla catla (Hamilton) and Oreachromis mossambicus (Peters), using Bkm 2(8) and M13 multilocus probes. Bkm 2(8) gave a higher number of bands when compared with M13. However, the number of bands obtained by each probe in O. mossambicus was similar. The higher band-sharing coefficient observed in this species may be attributed to inbreeding as it arose from a small founder population. In L rohita and C. catla, the Bkm 2(8) detected similar DNA fingerprints when two enzymes Hinfi and Taqi were used. The M13 probe also gave similar fingerprints with three restriction enzymes (Hinfi, Taqi, Alui). Comparison of the DNA fingerprints obtained by Bkm 2(8) and Ml 3 showed that these two probes detected different alleles. The overall similarity of the DNA fingerprint patterns in L. rohita and C. catla may be due to their genetic closeness as indicated by their same chromosome number, C-value and their ability to produce fertile hybrids. A similar argument also holds true for the Oreochromis species where interspecies hybridization results in fertile offspring.

Identification and Characterization of a Karyotypic Mutation inMagnaporthe grisea

We have identified karyotypic mutants of Magnaporthe grisea isolate Inal68. The mutants have a karyotype with an extra band of chromosome-ν in addition to six bands of the normal karyotype in contour-clamped homogeneous electric field (CHEF) electrophoresis. We named a karyotype of the original isolate as α′, and that of mutants as α′. The mutation has not affected the DNA fingerprint pattern, host specific pathogenicity (pathogenic race), or crossing abilities with the isolate Guy11. Karyotypic analyses of isolates from a repetitive single-spore isolation indicated that the frequency of mutation was 12.5% and that the mutation was limited in the manner of a to α′, and never reverted. Hybridization analyses showed that chromosome-ν originated from the chromosome in band IIIb of type α and might be produced by deletion.

DNA Fingerprinting Analysis of Native and Red Jungle Fowls in Fiji and Western Samoa.

In the present study, genomic DNA was prepared from dried chicken blood sample and subsequently used for analyzing DNA fingerprinting (DFP) of native and Red jungle fowl in the South Pacific Islands. These chicken genomic DNAs were sufficient in quantity and purity for multiple DFP analyses, and chicken blood could be stored for up to 3 months without physical degradation under dry condition.The DFP analyses were carried out by individual and population DFP. Similarities of DFP patterns and genetic distances among fowls in Fiji and Western Samoa were estimated using band sharing (BS) value and the mean number of nucleotide substitutions per nucleotide site, respectively. The results of population DFP were under the influence of data in individual DFP. From the BS values and genetic distances, the native fowls in five different islands of Fiji and Western Samoa could be distinguished in each island, and the difference was consistent with geographical distribution. Fijian and Western Samoan native fowls were found to form two separate groups. The Red jungle fowls in Fiji islands were closely related to each other, but were found to be related remotely to Red jungle fowls in Kagoshima University. Therefore, DFP analysis can be used to estimate relative genetic variability and to reconstruct the evolutionary relationships in small populations genetically isolated.

AFLP Analysis of Gene Pools of a Wild Bean Core Collection

Studies of diversity permit conclusions about the genetic structure of species. Systematically developed core collections are a particularly good tool for such purposes. The objective of this study was to evaluate the genetic structure between and within gene pools of a core collection of wild Phaseolus vulgaris L. using the technique of amplified restriction polymorphism (AFLP) analysis. DNA fingerprint patterns of 114 genotypes were analyzed by the NTSYS program. Major groups or gene pools were recognized in Mesoamerica, Colombia, the northern Andes of Ecuador and northern Peru, and the southern Andes, although the separation among gene pools was not wide. A unique segment of germplasm from northern Peru showed greater genetic distance than the other gene pools. Although most Mexican wild beans formed a rather homogenous group, a small number of accessions clustered with the Andean gene pool, and accessions from Guatemala tended to cluster apart from Mexican accessions. In the southern Andean gene pool, more discrete groups were formed which were associated with certain regions such as Argentina, possibly reflecting greater geographic and genetic isolation. Colombian accessions appeared to be highly introgressed with germplasm from other areas. AFLP analysis produced a large amount of data in a short period, thus permitting greater insights into the genetic structure of wild beans than had been possible with other methods of analysis.

Random amplified polymorphic DNA (RAPD) markers reveal genetic homogeneity in the endangered Himalayan species Meconopsis paniculata and M. simplicifolia

Random amplified polymorphic DNA (RAPD) marker-based analysis was carried out to study the extent of genetic polymorphism between populations of the two endangered Himalayan poppy species, Meconopsis paniculata and M. Simplicifolia. Of the 90 primers tested, 38 revealed marked inter-species genetic polymorphism between individuals of the two species from geographically isolated populations. However, intra-species genetic homogeneity was also evident with respect to a number of primers both within and between populations. A comprehensive analysis incorporating data from RAPDs, DNA fingerprinting and isozyme pattern was carried out and, based on the presence or absence of bands, three matrices of similarity indices were estimated. These matrices were subsequently utilized in cluster analysis. In order to compare the three clusters generated using these three different marker systems, a Mantel matrix-correspondence test was carried out on the basis of comparisons of co-phenetic values. The overall representation of relationships by cluster analysis was similar for all three marker systems and this was substantiated by high correlations among the three analyses revealed by the Mantel matrix-correspondence test. Our results point to very low or absence of, genetic polymorphism in M. paniculata and M. simplicifolia, and are in broad agreement with our previous observations on genetic diversity of Meconopsis species which point to a genetic basis for the possible extinction of this economically important genus.

The differentiation of Bordetella parapertussis and Bordetella bronchiseptica from humans and animals as determined by DNA polymorphism mediated by two different insertion sequence elements suggests their phylogenetic relationship.

We describe a novel insertion sequence (IS) element, IS1002, which was found to be closely related to IS481, which is found only in Bordetella pertussis; we found that these two IS elements have a level of sequence identity of 61.5% and also have almost identical terminal inverted repeats. IS1002 was present in both B. pertussis and Bordetella parapertussis strains isolated from humans. In contrast, IS1002 was absent from B. parapertussis strains isolated from sheep. A DNA fingerprint analysis performed with another IS element, IS1001, which is present in B. parapertussis and Bordetella bronchiseptica, revealed that B. parapertussis isolates obtained from sheep are distinct from human isolates. Thus, human and ovine B. parapertussis strains comprise two distinct populations, indicating that little or no transmission occurs between sheep and humans. An IS-associated restriction fragment length polymorphism analysis revealed by B. parapertussis strains isolated from sheep are genetically more polymorphic than the human B. parapertussis population, which is genetically very homogeneous. This suggests that human B. parapertussis strains diverged from a single clone only recently. IS1001 is present in a subset of B. bronchiseptica strains that were derived mainly from pigs and rabbits, suggesting that these strains had a common ancestry. On the basis of the results of a comparison of IS1001 band patterns and IS1001 sequences, ovine and human B. parapertussis strains appear to have evolved independently from B. bronchiseptica strains and to have adapted to different hosts (sheep and humans). Once in the human host, B. Parapertussis probably acquired IS1002 from B. pertussis. In contrast to human B. parapertussis isolates, B. pertussis strains produced polymorphic IS1002-related DNA fingerprint patterns.

Application of Randomly Amplified Polymorphic DNA Fingerprinting for Species Identification of Bacteria Isolated from Bovine Milk

A polymerase chain reaction-based DNA fingerprinting system for species identification of bacteria in milk was developed using randomly amplified polymorphic DNA. A total of 108 organisms including 24 ATCC reference strains and 84 wild-type isolates belonging to gram-negative, Staphylococcus, Enterococcus, and Streptococcus species were used to develop the system. Organisms included in the study were those that are isolated frequently from milk. Forty primers from two commercially available primer kits were evaluated to determine the “ideal” primer that could be used for several bacterial species. Over 960 DNA fingerprint patterns were analyzed by laser densitometry. Seven of the 40 primers met criteria established for primer selection. However, only primers OPE-4 (5′ GTGACATGCC-3′) and OPE-20 (5′-AACGGTGACC-3′) allowed differentiation between all 19 ATCC bacterial species included in the study. The other five primers were restricted to either gram-negative bacteria (OPA-7, OPA-14), Staphylococcus species (OPA-13, OPA-14, OPA-18), or Streptococcus species (OPA-3). Primers OPE-4 and OPE-20 were further evaluated using 84 wild-type isolates. A bacterial species identification scheme was developed based on characteristic polymorphic DNA fragments obtained with primers OPE-4 and OPE-20. Results of this study suggest that RAPD fingerprinting has the potential for being developed into a rapid and accurate method for species identification of bacteria in milk.

Differentiation of Xanthomonas albilineans strains with monoclonal antibody reaction patterns and DNA fingerprints

Thirty‐eight strains of the sugarcane leaf scald pathogen, Xanthomonas albilineans, were compared using a series of seven species‐specific monoclonal antibodies as well as genomic DNA fingerprint patterns. The strains, which were obtained from 13 countries and states in the USA, were placed in three major groups with approximately eight subgroups based on similarity of their serological reactions. The serological groupings correlated strongly with groupings based on DNA fingerprint patterns. The groups of strains that were genetically and serologically related did not necessarily come from nearby locations but were usually from widely separated regions. Thus, it appears that disease transmission occurred by means other than natural spread. The methods used in this study are useful for comparing relationships among strains of X. albilineans. A combination of the monoclonal antibodies could be used in a serodiagnostic‐based method to test for the presence of leaf scald disease.

SINE sequences detect DNA fingerprints in salmonid fishes

DNA probes homologous to two previously described salmonid short interspersed nuclear elements (SINEs) detected DNA fingerprint patterns in 14 species of salmonid fishes. The probes showed more homology to some species than to others and little homology to three nonsalmonid fishes. The DNA fingerprint patterns derived from the SINE probes are individual-specific and inherited in a Mendelian manner. Probes derived from different regions of the same SINE detect only partially overlapping banding patterns, reflecting a more complex SINE structure than has been previously reported. Like the human Alu sequence, the SINEs found in salmonids could provide useful genetic markers and primer sites for PCR-based techniques. These elements may be more desirable for some applications than traditional DNA fingerprinting probes that detect tandemly repeated arrays.

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.

EPIDEMIOLOGY AND MOLECULAR IDENTIFICATION OF SALMONELLA INFECTIONS IN CHILDREN. 1093

Human epidemics of Salmonella are commonly attributed to the ingestion of contaminated foods, but the role of foods in the development of disease in infants and children is not understood. The purpose of this study was to delineate the role of foods and the surrounding environment in the development of infections with Salmonella in infants and children. The families of children < 4 years of age who were infected withSalmonella between July 1993-June 1995 were given an extensive questionnaire about exposures, food preparation and consumption, pets in the home, and day care attendance. Three age-matched controls per patient were also queried. The two commonly isolated serotypes of Salmonella underwent restriction enzyme digestion (SpeI, XbaI) followed by pulsedfield gel electrophoresis in order to establish DNA fingerprint patterns. These patterns were visually inspected for similarities. Ninety cases and 264 controls were evaluable at the completion of the study. Univariate analysis of data demonstrated that controls rather than cases were more likely to have consumed food considered to be high risk (e.g., cole slaw, whole milk, uncooked apples, turkey; p<0.05), receive support from WIC (p<0.02) and Medicaid (p<0.01), and boiled their water prior to mixing formula(p<0.05). S. newport and S. typhimurium were the most common isolates recovered (62%) and the DNA fingerprinting data from these isolates demonstrated 23 unique patterns for S. newport and 21 forS. typhimurium. Identical fingerprint patterns were found in 4 fingerprint clusters involving 10 patients with S. newport and in one cluster involving 2 patients with S. typhimurium. Review of the demographic data demonstrated that 7 of the children had a history of living or traveling to the same town as others with identical isolates. Although the majority of cases of salmonellosis in children encountered were sporadic, molecular fingerprinting demonstrated that at least 14% of our cases were infected with isolates identical to that of another child. The lack of common epidemiologic factors implicating contaminated foods suggests that environmental contamination remains important in the development of salmonellosis in infants and children. Until the role of environmental contamination is understood, prevention of this disease will not occur.

CAC--the neglected repeat.

It is becoming increasingly clear that repetitive DNA is of biological significance as well as experimental importance. Here we review the information available about one type of repetitive DNA, the trinucleotide repeat (CAC)n, and briefly compare it with other trinucleotide repeats. Although much work has been done in analysing DNA fingerprinting patterns produced using the synthetic oligonucleotide (CAC)5 as a probe, there is relatively little information about individual (CAC)n-containing sequences and their abundance, organisation and distribution in mammalian DNA. From the data that is available, it is clear that there are at least two areas that should repay further study: (1) the organisation and generation of long sequences that contain (CAC)n motifs as part of a larger repeating unit (minisatellites) and (2) the distribution of small (CAC)n sequences (microsatellites), in particular their relationship to genes.

Molecular epidemiology of tuberculosis in Denmark in 1992.

The incidence of tuberculosis (TB) is increasing all over the world, including in countries with a high standard of living and good social security. Denmark represents such a region. Furthermore, it is a small country (5 million inhabitants) with a long tradition in TB control, including a centralization of the bacteriological diagnostic facility. The present study was intended to analyze the transmission of Mycobacterium tuberculosis in a country in which TB has low endemicity by a combination of conventional epidemiological approaches and DNA fingerprinting techniques, whereby individual bacterial strains can be traced. M. tuberculosis isolates from 92% of all new cases of bacteriologically verified TB in Denmark during 1992 were subjected to IS6110 DNA fingerprinting to visualize the DNA restriction fragment length polymorphism (RFLP) patterns of the isolated strains. The data obtained from the RFLP analyses were interpreted by using demographic data, such as age, sex, ethnicity, and residence, for the patients. The risk factors among the patients for being part of an active chain of transmission, as opposed to demonstrating reactivation of a previously acquired latent infection, were estimated by statistical analyses. The magnitude of TB transmission in 1992 in Denmark was determined, and transmitted infections were shown to comprise at least one quarter of the total number of cases. Almost half of the TB cases involved patients of foreign origin. However, most of these isolates showed unique DNA fingerprint patterns and were rarely part of an active chain of transmission. The major chains of recent transmission were localized to distinct geographical regions in the country. TB is frequent among immigrants, especially from Asia and Africa, but it is apparently readily suspected, diagnosed, and treated by the health care system. Danish patients with pulmonary symptoms are not primarily suspected to have TB and, therefore, play an important role in recent TB transmission in Denmark.