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PubMed1996

Relationship between band sharing levels of DNA fingerprints and inbreeding coefficients and estimation of true inbreeding in turkey lines.

A regression analysis between band sharing of DNA fingerprints and calculated inbreeding coefficients was conducted using six experimental turkey lines. The DNA fingerprints were produced from 18 individual DNA samples per line representing different families. The DNA was digested with a HaeIII restriction enzyme and hybridized with Jeffreys' 33.6 probe. The band sharing within lines ranged from 0.42 to 0.62. The inbreeding coefficients of the lines were calculated based on population sizes and variation in family sizes. The inbreeding coefficients varied from 2.5 to 45%. Regression analysis between the two variables yielded a highly significant (P < or = 0.0001) linear model with a correlation coefficient of 0.992. The linear model was used to estimate the actual inbreeding in these lines.

Animals↗
PubMed1997

Fingerprinting of Ehrlichia species by repetitive element polymerase chain reaction.

To facilitate identification of ehrlichial pathogens, we developed a new technique based on fingerprints resulting from repetitive element polymerase chain reaction (rep-PCR). This technique uses consensus tRNA primers to generate amplification products that reflect distance polymorphisms between adjacent tRNA genes. Species-specific fingerprint patterns were obtained for seven Ehrlichia spp., as well as the unnamed causative agent of human granulocytotropic ehrlichiosis. Bands ranged in size from approximately 50 to 1,000 base pairs. Banding patterns varied depending on dilution of template DNA, with lower dilutions giving more complex banding patterns. These preliminary data indicate that repetitive-sequence-based PCR appears to be a useful technique for identifying ehrlichial organisms to the species, and perhaps the strain level. Compared with other conventional molecular-biologic methods, rep-PCR offers the advantages of ease of performance and rapid availability of results.

Bacterial Typing Techniques↗
PubMed2001

Quantifying mosquito biting patterns on humans by DNA fingerprinting of bloodmeals.

A major debate in infectious disease epidemiology concerns the relative importance of exposure and host factors, such as sex and acquired immunity, in determining observed age patterns of parasitic infection in endemic communities. Nonhomogeneous contact between hosts and vectors is also expected to increase the reproductive rate, and hence transmission, of mosquito-borne infections. Resolution of these questions for human parasitic diseases has been frustrated by the lack of a quantitative tool for quantifying the exposure rate of people in communities. Here, we show that the polymerase chain reaction (PCR) technique for amplifying and fingerprinting human DNA from mosquito bloodmeals can address this problem for mosquito-borne diseases. Analysis of parallel human and mosquito (resting Culex quinquefasciatus) samples from the same households in an urban endemic focus for bancroftian filariasis in South India demonstrates that a 9-locus radioactive short-tandem repeat system is able to identify the source of human DNA within the bloodmeals of nearly 80% of mosquitoes. The results show that a person's exposure rate, and hence the age and sex patterns of exposure to bites in an endemic community, can be successfully quantified by this method. Out of 276 bloodmeal PCR fingerprints, we also found that on average, 27% of the mosquitoes caught resting within individual households had fed on people outside the household. Additionally, 13% of mosquitoes biting within households contained blood from at least 2 people, with the rate of multiple feeding depending on the density of humans in the household. These complex vector feeding behaviors may partly account for the discrepancies in estimates of the infection rates of mosquito-borne diseases calculated parasitologically and entomologically, and they underline the potential of this tool for investigating the transmission dynamics of infection.

Adolescent↗
PubMed2004

Practical application of DNA fingerprinting to trace beef.

DNA fingerprinting allows the verification of conventional methods used to implement beef traceability. At any point along the supply chain, the identity of an animal or piece of meat can be checked by comparison of its DNA profile with an initial sample. Practical application of DNA fingerprinting to trace beef requires a choice of DNA markers as well as the optimization of sampling methods. This has been achieved as the result of collaboration between meat technicians and geneticists over a period of 4 years. The discrimination power of nine highly polymorphic microsatellite markers was evaluated. We propose that three markers (with a 0.001 probability that two individual profiles match by chance) are adequate for routine tests. Two key points along the production-commercialization chain where sampling must be systematic were defined: (i) the tagging of the calf (identity control) and (ii) after slaughter (slaughter control), before the animal loses its external appearance. The identity control was blood collected on a filter paper adapted to the ear tag; the slaughter control was the tagged ear itself. These constituted the control samples, which were archived with a code matching the individual tag number. Test samples were obtained on a random basis from live animals, carcasses, and pieces of meat at cutting halls and at the retail outlet and in cases when the verification of identity was needed. The DNA profiles of the test samples and the controls were then obtained and compared, to verify either an individual identity or the origin of a piece of meat from the stated animal.

Animal Identification Systems↗
PubMed1999

Application of restriction fragment fingerprinting with a rice microsatellite sequence to assembling rice YAC clones.

To refine the current physical map of rice, we have established a restriction fragment fingerprinting method for identifying overlap between pairs of rice yeast artificial chromosome (YAC) clones and defining the physical arrangement of YACs within contiguous fragments (contigs). In this method, Southern blots of rice YAC DNAs digested with a restriction endonuclease are probed with a rice microsatellite probe, (GGC)5. The probe produces a unique fingerprint profile characteristic of each YAC clone. The profile is then digitized, processed in a computer, and a statistic that represents the degree of overlap between two YACs is calculated. The statistics have been used to detect overlaps among YAC clones, thereby filling a gap between two neighbouring contigs and organizing overlapping rice YAC clones into contiguous fragments. We applied this method to rearranging YACs that had previously been assigned to rice chromosome 6 by anchoring with RFLP markers.

Chromosomes, Artificial, Yeast↗
PubMed1999

Estimation of serial interval and incubation period of tuberculosis using DNA fingerprinting.

OBJECTIVE: To determine the frequency distributions of serial interval and incubation period of tuberculosis within 4 years of transmission, and to identify correlates of serial intervals and incubation periods. METHODS: DNA fingerprints were obtained for all isolates from all culture-positive patients notified in The Netherlands from 1993 to 1996. Patient information was obtained from the National Tuberculosis Register. Results from contact investigations were provided by public health services. Source cases and secondary cases of tuberculosis were identified, based on 1) identical DNA fingerprints, and 2) epidemiological confirmation of contact. Under-representation of long intervals were corrected for by weighting cases. RESULTS: A total of 69 source-secondary case couples were identified. The geometric mean serial interval was 29.5 weeks (95% confidence interval [CI] 22.8-38.2 weeks) and the geometric mean incubation period 20.8 weeks (95% CI 15.5-27.8 weeks). Serial intervals and incubation periods tended to increase with age (P > 0.05). Three secondary cases with human immunodeficiency virus infection showed very short incubation periods (P > 0.05). CONCLUSION: Using a new methodology, the distribution of incubation periods of tuberculosis gave results consistent with earlier studies.

Adolescent↗
PubMed1999

[Child with tubercular meningoencephalitis, etiologically related to a microepidemic of tuberculosis in Southern Limburg by DNA 'fingerprinting'].

UNLABELLED: MICROEPIDEMIC: In a child fatal tuberculous meningoencephalitis was diagnosed and the Regional Public Health Service Geleen, Limburg, the Netherlands, was notified. Source identification and contact tracing (ring investigation) did not reveal a source of the infection. In a person living in the same village pulmonary tuberculosis had been diagnosed, but there was no evident contact between both patients. When the mycobacteria from all patients with tuberculosis were typed by DNA fingerprinting, both patients belonged to the same cluster, thus identifying the infection source of the meningoencephalitis patient. In the management of the outbreak 950 persons were examined in a contact tracing survey. Of them 35 had recently been infected and four of these had recently acquired pulmonary tuberculosis. DISCUSSION: Highly infectious patients with tuberculosis are able to infect persons who cannot be found by conventional contact tracing survey as the transmission of tuberculosis is more subject to casual encounters than was hitherto believed. DNA fingerprinting is a very useful method in contact investigation of tuberculosis. In the Netherlands, therefore, the early diagnosis and treatment of symptomatic patients with infectious tuberculosis is more important to stop the transmission of Mycobacterium tuberculosis than the identification and screening of risk groups in the population.

Adult↗
PubMed1999

[The use of molecular fingerprinting techniques in environmental hygiene].

The analysis of epidemiological pathways for the transmission of pathogens via the environment is a fundamental task in the field of environmental hygiene. The task demands for a precise differentiation between isolates. This can be done by sophisticated techniques for differentiation which are based on phenotypic or genotypic features. The fingerprinting of DNA is a powerful tool in this context. A survey is given for different kinds of RFLP-analysis, plasmid profiling, and PCR fingerprinting as well regarding their principles, methodical variations and published applications. Notable limitations of the applications are also discussed.

Animal Diseases↗
PubMed1998

[Study on the molecular epidemiology characteristics of Pseudomonas cocovenenans subsp. farinofermentans isolated from China with rDNA fingerprinting].

After the chromosomal DNA digestion by Bgl II and EcoRV, rDNA fingerprinting for Pseudomonas cocovenenans subsp. farinofermentans was made using biotin-labeled 16S + 23S rDNA as a probe. Fifty one strains of P. cocovenenans subsp. farinofermentans isolated from some areas of China were divided into 20 ribotyes, 52.9 percent of which were RT6, 9 and 2. In most cases, there were 2 or more ribotypes in each province or city. Though same ribotypes were isolated from different provinces, each ribotype existed in part of an area. The ribotype of RT6 was detected in samples from 4 incidents of food poisoning in Guizhou, Jiangsu and Hebei provinces, which indicated that these incidents may be caused by the same clone. With discrimination and cluster analysis of ribotypes, the rDNA fingerprinting may effectively contribute to analyzing the homogeneity of different strains, tracing the transmission route, and exploring the evolution and epidemiological characteristics of P. cocovenenans subsp. farinofermentans.

China↗
PubMed1999

Efficient DNA fingerprinting method for the identification of cross-culture contamination of cell lines.

In order to identify cross-culture contamination of cell lines, we applied DNA fingerprinting using variable number of tandem repeat (VNTR) loci and short tandem repeat (STR) loci amplified by polymerase chain reaction (PCR) instead of a radioisotope labeled multilocus probe. Eleven cell lines were used for the Apo B and D1S80 loci detection, and twelve cell lines were examined in the Y-chromosome analysis. The data obtained from the sister cell lines NALM-6 and B85, two MOLM-1 cultures from two cryopreserved tubes, and four subclones of BALM-9 and its sister cell line BALM-10, displayed clear and distinct bands of each PCR product for both Apo B and D1S80. Detection of a Y-chromosome DNA sequence is another very informative marker for the identification of cell lines, if the Y-chromosome is present. We examined eight cell lines for the expression of four STR loci; the data thus generated were compared with the results previously reported from other laboratories. The resulting electrophoretic banding patterns showed that our "home-made" STR detection system is a useful and efficient tool for the authentication of cell lines. PCR detection of VNTR and STR loci represents a simple, rapid and powerful DNA fingerprinting technique to authenticate human cell lines and to detect cross-culture contamination. This PCR technique may be used in lieu of the more time-consuming, labor-intensive and radioactive Southern blot multilocus method.

Base Sequence↗
PubMed2000

Male-specific band in random amplified microsatellite polymorphism fingerprints of Holstein cattle.

A total of 65 blood samples collected from Holstein cattle were employed for DNA extraction. Genomic DNA were amplified by means of random amplified polymorphic DNA (RAPD). One hundred and one random primers (Operon kits OPAA, OPAO, OPAV, OPC, OPE and OPA-06) were used for polymerase chain reactions (PCR). The PCR products were size fractionated by means of electrophoresis in agarose gel, transferred to a nylon membrane, and then hybridized with a alpha-32P-labeled microsatellite probe for random amplified microsatellite polymorphism (RAMPO) analysis. All the primers produced many amplification patterns in RAMPO fingerprints, but only OPE-01 (5'-CCCAAGGTCC-3') yielded a male-specific strong signal. Among the 65 cases, the specific band could be observed in the RAMPO fingerprints of the 24 male samples but not in the 41 female samples. Therefore, this specific band is peculiar to male cattle.

Animals↗
PubMed2000

Using DNA fingerprinting to detect transmission of Mycobacterium tuberculosis among AIDS patients in two health-care facilities in Puerto Rico.

BACKGROUND: Fourteen cases of tuberculosis (TB) in Puerto Rico, diagnosed from April 1993 to April 1995, had the same DNA fingerprint, documenting disease caused by the same strain of Mycobacterium tuberculosis. The 14 cases were retrospectively investigated for epidemiologic links. METHODS: Records were reviewed and staffs of the TB program, hospital/clinic, and AIDS residential facilities were interviewed. RESULTS: Half of the AIDS cases were epidemiologically related, providing evidence of TB transmission in an emergency department, an AIDS inpatient ward, and an AIDS residential facility. DNA fingerprinting allowed detection of M tuberculosis transmission, but contact investigators could have documented it sooner. Factors contributing to transmission included delayed diagnosis, prolonged infectiousness, inadequate discharge planning and infection control procedures, and poor communication between health-care facilities. CONCLUSIONS: The numbers of AIDS residential facilities are increasing and must understand proper monitoring of TB patients and infection control measures that prevent transmissions.

AIDS-Related Opportunistic Infections↗
PubMed2000

[Concerning cell cultures for biocompatibility-testing: monitoring by DNA-fingerprinting].

Cell cultures are innovative tools for e.g. biocompatibility testing of biomaterials in vitro. In our studies we used fibroblast, endothelial cell and chondrocyte cultures of human origin and of the test animal species most common for this purpose in vivo. Verification of the identity of these cells is obligatory for reproducibility of the tests and valid interpretation of the results. Cultured cells have to be checked for identity, contaminations of various origins and also for genomic mutations occuring during prolonged cultivation in vitro or due to exposition to biomaterials. Furthermore, the risk of genetic cross-contamination with other cells increases with the number of cell cultures passaged parallel in the same laboratory. Therefore, we generated reference fingerprints of the cultures in varying passages for comparative monitoring of cells purposed for in vitro tests. Minisattelite DNA polymorhism resulting in reproducible individual DNA fingerprints is very discriminatory and can be used for cell culture monitoring. The patterns are stable over several passages, although sudden changes did happen in two cases, i.e. loss/gain of bands or changes in band-intensity, indicating massive genomic mutations of the cultures in vitro. Influences of biomaterials on the prints could not be detected. Several tasks can be followed at the same time: detection of contaminant cells, identification of these cells of primary culture origin used for in vitro testing and finally, monitoring for eventual genomic mutations due to prolonged cultivation or contact to biomaterials. Inconclusive results in just one of these aspects should lead to the disqualification of the monitored cultures from usage in vitro.

Animal Testing Alternatives↗
PubMedDate not supplied

[Study of genome instability using DNA fingerprinting of the offspring of male mice subjected to chronic low dose gamma irradiation].

By a polymerase chain reaction with an arbitrary primer (AP-PCR), the possibility of transmission of genome instability to somatic cells of the offspring (F1 generation) from male parents of mice exposed to chronic low-level gamma-radiation was studied. Male BALB/c mice 15 days after exposure to 10-50 cGy were mated with unirradiated females. Biopsies were taken from tale tips of two month-old offspring mice and DNA was isolated. The primer in the AP-PCR was a 20-mer oligonucleotide flanking the microsatellite locus Atp1b2 on chromosome 11 of the mouse. A comparative analysis of individual fingerprints of AP-PCR products on DNA-templates from the offspring of irradiated and unirradiated male mice revealed an increased variability of microsatellite-associated sequences in the genome of the offspring of the males exposed to 25 and 50 cGy. The DNA-fingerprints of the offspring of male mice exposed to chronic irradiation with the doses 10 and 25 cGy 15 days before fertilization (at the post-meiotic stage of spermatogenesis) showed an increased frequency of "non-parent bands". The results of the study point to the possibility of transmission to the offspring somatic cells of changes increasing genome instability from male parents exposed to chronic low-level radiation prior to fertilization.

Animals↗
PubMed2001

The effect of age and study duration on the relationship between 'clustering' of DNA fingerprint patterns and the proportion of tuberculosis disease attributable to recent transmission.

Though it is recognized that the extent of 'clustering' of isolates from tuberculosis cases in a given population is related to the amount of disease attributable to recent transmission, the relationship between the two statistics is poorly understood. Given age-dependent risks of disease and the fact that a long study (e.g. spanning several years) is more likely to identify transmission-linked cases than a shorter study, both measures, and thus the relationship between them, probably depend strongly on the ages of the cases ascertained and study duration. The contribution of these factors is explored in this paper using an age-structured model which describes the introduction and transmission of M. tuberculosis strains with different DNA fingerprint patterns in The Netherlands during this century, assuming that the number of individuals contacted by each case varies between cases and that DNA fingerprint patterns change over time through random mutations, as observed in several studies. Model predictions of clustering in different age groups and over different time periods between 1993 and 1997 compare well against those observed. According to the model, the proportion of young cases with onset in a given time period who were 'clustered' underestimated the proportion of disease attributable to recent transmission in this age group (by up to 25% in males); for older individuals, clustering overestimated this proportion. These under- and overestimates decreased and increased respectively as the time period over which the cases were ascertained increased. These results have important implications for the interpretation of estimates of the proportion of disease attributable to recent transmission, based on 'clustering' statistics, as are being derived from studies of the molecular epidemiology of tuberculosis in many populations.

Adolescent↗
PubMed2001

DNA fingerprinting of methicillin-resistant Staphylococcus aureus by pulsed-field gel electrophoresis (PFGE): comparison of strains from 2 Malaysian hospitals.

AIM OF STUDY: To determine and compare the pulsed-field gel electrophoresis (PFGE) patterns of endemic MRSA strains in 2 major Malaysian hospitals and to compare the PFGE patterns with antibiotypes of the strains studied. METHODS: Fifty-six MRSA strains selected randomly between September 1997 and July 1998 from Hospital Queen Elizabeth (HQE) and Hospital Umum Sarawak (HUS) were tested for antimicrobial resistance and DNA fingerprinting was carried out by pulsed-field gel electrophoresis (PFGE) technique. RESULTS: Seven PFGE types were recognised (A, B, C, D, E and F). All 7 PFGE types were observed in HQE while only 2 PFGE types (B, C) were noted in HUS strains. There is a predominance of a single PFGE pattern (type B) in both hospitals, as seen in 46% of HQE strains and 89% of HUS strains. Subtype B2 was the commonest subtype in HQE while subtype B1 predominated in HUS. Strains resistant to fusidic acid and rifampicin exhibited PFGE type F that is unique to HQE. All strains were resistant to penicillin, erythromycin, cotrimoxazole, tetracycline and gentamicin. Strains with the same antibiotic susceptibility pattern can be different PFGE types. CONCLUSION: Molecular typing of the MRSA by PFGE is a useful tool in the study of endemic strains present in an institution. Strains in HQE were found to be more heterogeneous than HUS strains. Common PFGE types can also be seen in both hospitals suggesting that some of the strains was genetically related and has propagated within and between the 2 hospitals. Our findings also indicate that the relationship between antibiotic susceptibility and PFGE patterns was not close and antibiograms should not be relied upon for typing strains in epidemiological studies. By knowing the DNA fingerprints of the isolates endemic in each hospital, the spread of MRSA with a particular PFGE type can be monitored within and between hospitals.

DNA Fingerprinting↗
PubMed2001

Investigation of tuberculosis transmission in Canadian Arctic Inuit communities using DNA fingerprinting.

OBJECTIVE: To understand the transmission of tuberculosis in Inuit communities in the Baffin region of the Canadian Arctic. METHODS: Twenty-one isolates of Mycobacterium tuberculosis from 19 Inuit patients diagnosed with tuberculosis between February 1991 and September 1993 were analyzed by DNA fingerprinting. The DNA fingerprints were achieved by the standard restriction fragment length polymorphism (RFLP) technique, with subsequent probing using the repetitive insertion segment IS6110. RESULTS: The isolates could be divided into three DNA types. The DNA types generally corresponded to the geographic origins of the patients. In most instances only one DNA type of M. tuberculosis was identified in each community. This suggests that a single case was the start of each of the three clusters, most likely due to reactivation. CONCLUSIONS: The results show that molecular typing of M. tuberculosis was useful in determining the mode of transmission of tuberculosis in a remote area of the Canadian Arctic where the disease is endemic. In addition, the information provides useful information for planning interventions in this setting.

Adolescent↗
PubMed2001

Clonal relations among Salmonella enteritidis phage type 3 outbreak isolates traced by DNA fingerprinting.

Isolates of Salmonella enteritidis PT3, a rare phage type, were recovered from patients and strains were isolated from an outbreak of gastroenteritis that occurred during the summer of 1997 in North-East Sardinia, Italy. To investigate possible clonal involvement in the outbreak and to evaluate the capacity to discriminate among S. enteritidis PT3 strains, a number of molecular typing methods including ribotyping with a mixture of PstI and SphI (PS-ribotyping), PFGE with endonuclease XbaI and RAPD typing with four arbitrary primers was used. The typical XbaI endonuclease generated PFGE pattern also explained the prevalence of highly clonal S. enteritidis PT3 strains in the outbreak and adjacent areas. RAPD fingerprinting with primers OPA 4, OPB 15, OPB17 and P1254 exhibited a single but unique RAPD profile among the outbreak strains from various sources that differed significantly from control strains. The results of this study showed that when an appropriately chosen set of primers is employed, RAPD fingerprinting can be used as an alternative, rapid, highly reproducible technique for tracing the clonal relations of S. enteritidis PT3, and can be more discriminatory than PFGE. Furthermore, this study revealed the possibility of PT3 causing outbreak.

DNA Fingerprinting↗