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At least 199 records · Page 11Linked to original sources

PubMed1994

Isolation of a Rhizobium galegae strain-specific DNA probe.

We have isolated a strain-specific DNA probe from the strain Rhizobium galegae HAMBI 1174 by a subtraction hybridization procedure followed by PCR amplification and DNA cloning. The specificity of the 342-bp DNA probe (P3) was tested in dot blot or Southern blot hybridizations against total genomic DNA of 41 bacterial strains (21 of them belong to R. galegae, 15 to other Rhizobium species and five to other bacterial species). Only the samples from four R. galegae strains, which are different isolates but identical to the strain HAMBI 1174, hybridized with the probe. The P3 probe was sequenced and PCR primers were designed based on its sequence. PCR amplification from purified total genomic DNA of 52 strains and subsequent hybridization with the P3 probe proved that the primers are strain specific.

Base Sequence↗

PubMed1995

Synthesis of PCR-derived, digoxigenin-labeled DNA probes for in situ detection of Epstein-Barr early RNAs in Epstein-Barr virus-infected cells.

A PCR-derived digoxigenin-labeled DNA probe was used for for Epstein-Barr early RNA (EBER) in situ hybridization in formalin-fixed paraffin-embedded tissues. The results showed that the hybridization signal was morphologically distinct and the intensity of signal was comparable with those by RNA riboprobe. The advantages of using PCR-derived DNA probes for EBER in situ hybridization include: (1) the synthesis of digoxigenin-labeled DNA probes is easy and simple by PCR; (2) the labeled amplification product can be used as a probe without further purification; (3) DNA probes are potentially more stable than RNA probes; and (4) the preparation of DNA probes is relatively efficient and rapid. It is concluded that this technique is an ideal candidate for detection of EBER expression in clinical specimens.

Base Sequence↗

PubMedDate not supplied

Production of digoxigenin-labelled parvovirus DNA probe by PCR.

A 560-bp digoxigenin(Dig)-labelled DNA-probe was produced by PCR using a 699-bp parvovirus DNA fragment as template with introduction of Dig-dTUP into the PCR reaction mixture. It was found to be very important to pay close attention to the amount of template employed, the number of cycles used, predenaturation of target DNA and optimization of the percentage of dTTP substituted by Dig-DUTP in the reaction mixture. The same 560-bp DNA fragment produced by PCR without the incorporation of Dig-DUTP in the reaction mixture, was subsequently labelled with Dig-dUTP by the random primed labelling method. Both of the Dig-labelled parvovirus DNA probes described above showed the same DNA detection level (about 1 pg), but production of the probe with Dig-DUTP incorporated in the PCR reaction mixture was much simpler.

Base Sequence↗

PubMed1988

Preparation and chromatographic use of 5'-fluorescent-labelled DNA probes.

A convenient procedure for synthesizing and purifying fluorescently-labelled short DNA probes is reported. DNA probes were chemically synthesized on an automated instrument using the "Aminolink" reagent in the final cycle to attach a primary amino group at the 5'-terminus in the final step. The synthetic oligonucleotides were purified by polyacrylamide urea gel electrophoresis, followed by reversed-phase high-performance liquid chromatography (HPLC). The oligomers were then allowed to react with a fluorescent compound, and the products were separated by HPLC with consecutive detection by UV absorption and fluorescence. Gel permeation chromatography demonstrated that the fluorescent probes were able to form stable hybrids with complementary oligodeoxynucleotides. Furthermore, essentially 100% of the purified fluorescent probe was capable of hybridizing to its complementary strand. Special precautions in handling the fluorescent probes, such as stability, were investigated.

Chromatography, High Pressure Liquid↗

PubMed2003

A rapid DNA probe test compared to culture methods for identification of subgingival plaque bacteria.

There has been a significant amount of interest in developing a more rapid and cost-effective test to identify bacterial pathogens in plaque. DNA probe technology may meet both these objectives, it is more rapid and cost-effective when compared to culture methods. The purpose of this study was to compare an automated DNA probe test with classical culture methods for identifying Bacteroides forsythus and Porphyromonas gingivalis in subgingival plaque of patients with adult periodontitis. Subgingival plaque samples were collected from sites with moderate to severe periodontitis and divided into two aliquots for analysis by either DNA probe or culture methods. When the DNA probe method was compared with the culture method (gold standard), the sensitivity and specificity for B. forsythus were 92.0% (SE = 3.4%) and 50.5% (SE = 7.8%), respectively; for P. gingivalis they were 52.2% (SE = 8.7%) and 74.7% (SE = 5.9%), respectively. Detection of B. forsythus and P. gingivalis by DNA probe correlated with probing depth (P = 0.01 for B. forsythus and P = 0.03 for P. gingivalis). It was concluded the DNA probe test was comparable to culture methods in detecting B. forsythus. In addition, when compared to the culture method, a better correlation was obtained with DNA probe detection of B. forsythus or P. gingivalis and clinical parameters.

Adult↗

PubMed1990

[Comparative study with digoxigenin and biotin markers for the DNA probe identification of genes coding for type TEM beta-lactamases].

We have tested two non-isotopic labels: digoxigenin--11-dUTP and biotin-7-dATP for the detection of TEM-1 beta-lactamase gene with a TEM-1 probe by DNA:DNA hybridisation (using spot technique). The use of radioactive labels is inconvenient and not available to all the clinical bacteriological laboratories. The strains tested were: 16 on file (14 carriers of different types of beta-lactamase) and 19 clinical isolated strains. Previously, the plasmid beta-lactamase of these strains had been identified by analytical isoelectric focusing. Using the probe labeled with digoxigenin-11-dUTP, the correlation between results obtained by DNA:DNA hybridisation and those by analytical isoelectric focusing was excellent, with no false positives; with the biotin--7-dATP labeled probe, reading was difficult and some false positives were observed.

Biotin↗

PubMed1989

The use of a DNA probe for epidemiological studies of candidiasis in immunocompromised hosts.

Reproducible typing procedures to differentiate isolates of Candida albicans are limited. C. albicans isolates were obtained from immunocompromised patients by using DNA restriction enzyme fragment analysis and hybridization with both a radiolabeled mitochondrial DNA probe and a nonradioactive (biotinylated) DNA probe. There were 110 pathogenic and nonpathogenic C. albicans isolates from 63 immunocompromised patients. EcoRI restriction fragment analysis with the biotinylated probe revealed different "fingerprint" patterns for 60 of 63 patients. Analysis of 57 isolates from 20 patients showed no intrapatient variation regardless of the isolation site. DNA probe "fingerprint" patterns were analyzed for eight patients on serially recovered (range, 2-18 mo) C. albicans isolates. The unique patient profiles persisted over time. The application of this biotinylated C. albicans DNA probe provides a more sensitive means than simple gel restriction fragment analysis to define the epidemiology of C. albicans infection. The use of this biotin-labeled nonradioactive probe has potential application in clinical evaluations of outbreaks of nosocomial candidiasis.

Biotin↗

PubMed1991

Preparation of europium-labelled DNA probes and their properties.

A chemical method for labelling DNA with a europium chelate is presented. First, primary aliphatic amino groups are introduced onto DNA in a transamination reaction. The transamination reaction is altered by adjusting temperature and duration of the reaction. Subsequently, the modified DNA is reacted with an isothiocyanate derivative of a Eu chelate. The optimum amount of Eu chelates on a DNA probe is 4-8% of total nucleotides. There is a decrease of 0.7 degrees C in the melting temperature of DNA for each incorporated Eu chelate on 100 bases. Hybridization efficiency is lowered by the introduction of Eu chelates but this effect can be partly overcome by using high DNA probe concentrations. The detection limit of the Eu-labelled probe is 0.15 attomoles of target DNA in a mixed-phase hybridization assay on microtitration wells. In addition to high sensitivity the Eu-labelled probes offer convenience in use and results which are quantitative and easy to interpret.

Affinity Labels↗

PubMed1988

Sexing whole human pre-embryos by in-situ hybridization with a Y-chromosome specific DNA probe.

We have used DNA-DNA in-situ hybridization with a DNA probe for the human Y-chromosome to distinguish between male and female human pre-embryos. Both biotinylated and tritiated Y-probes worked well on control cell cultures where 100 interphase nuclei were scored. Morphologically normal pre-embryos could be sexed with confidence with the tritiated Y-probe but the biotin results were less reliable (although only a few pre-embryos were analysed in this way). Early cleavage stage pre-embryos had large nuclei with relatively diffuse Y-bodies and were more difficult to score with the biotinylated Y-probe. Morphologically abnormal pre-embryos often had large nuclei with multiple Y-bodies (presumably polyploid nuclei) or small nuclei with no Y-bodies (possibly fragmenting nuclei). In all, 38 cleaving and two non-cleaving pre-embryos were analysed. The incidence of false positive and false negative cells seen after hybridization of tritiated Y-probes to control lymphocyte cultures suggests that it should normally be possible to distinguish morphologically normal male and female pre-embryos with samples of three to six interphase nuclei.

Blastocyst↗

PubMed1992

Comparison of a commercial DNA probe test and three cultivation procedures for detection of Mycobacterium paratuberculosis in bovine feces.

Diagnosis of paratuberculosis using the IDEXX DNA probe test and 3 methods for cultivation of Mycobacterium paratuberculosis from fecal specimens were compared. Twenty-one of 170 fecal specimens were DNA probe test positive, whereas 35 specimens were positive by 1 or more of the cultivation methods evaluated. Four specimens were DNA probe test positive but were negative by fecal culture. The probe test detected M. paratuberculosis DNA in 62.9% of the specimens positive by a sedimentation culture method, in 56.6% of those positive by a centrifugation culture method, and in 65.4% of the specimens positive by the Cornell culture method. Specificity of the DNA probe test was approximately 97% relative to all culture methods. Generally, the probe test detected M. paratuberculosis DNA in fecal specimens from animals shedding at least 10(4) M. paratuberculosis colony forming units per gram of feces. Although the probe test did not detect all of the cattle shedding M. paratuberculosis, it was possible to identify cattle shedding the greatest number of organisms in 3 days compared with a minimum of 6 weeks required for positive culture results. The centrifugation method resulted in the most isolations of M. paratuberculosis after 12 weeks of incubation. However, contamination also was greatest when the centrifugation method was used. Contamination was best controlled using the Cornell method. The sedimentation method was the least time consuming and yielded results similar to those of the other 2 methods.

Animals↗

PubMed2001

Rapid detection of Listeria monocytogenes in foods, by a combination of PCR and DNA probe.

Listeria monocytogenes is a frequent contaminant of water and foods. Its rapid detection is needed before some foods can be prepared for marketing. In this work L. monocytogenes has been searched for in foods, by a combination of polymerase chain reaction (PCR) and a DNA probe. Both PCR and the probe were prepared for recognizing a specific region of the internalin gene, which is responsible for the production of one of the most important pathogenic factors of Listeria. The combined use of PCR and the DNA probe was used for the detection of L. monocytogenes in over 180 environmental and food samples. Several detection methods were compared in this study, namely conventional culture methods; direct PCR; PCR after an enrichment step; a DNA probe alone; a DNA probe after enrichment and another commercially available gene-probe. Finally PCR and the DNA probe were used in series on all the samples collected. When the DNA probe was associated with the PCR, specific and accurate detection of listeria in the samples could be obtained in about a working-day. The present molecular method showed some advantages in terms of rapidity and specificity in comparison to the other aforementioned tests. In addition, it resulted as being easy to handle, even for non-specialized personnel in small diagnostic microbiology laboratories.

Aged↗

PubMed1993

Species-specific recombinant DNA probes for Mycoplasma meleagridis.

Two recombinant DNA probes (pMM-2 and pMM-13) were isolated from a Mycoplasma meleagridis strain 17529 genomic library prepared in plasmid pUC8, and Escherichia coli strain JM83. In dot blot assays, 32P-labeled pMM-13 with a DNA insert of 3.5 kbp, hybridized with 18 isolates of M. meleagridis but not with 16 other known species of avian mycoplasmas. Except for weaker signals on hybridization with the M. meleagridis cultures, pMM-2 with an DNA insert of 0.85 kbp, showed a similar reaction pattern. The minimal concentration of M. meleagridis strain 17529 chromosomal DNA that pMM-13 and pMM-2 detected were 1 and 8 ng, respectively. Neither probe hybridized with chromosomal DNA of M. gallisepticum strain S6, M. synoviae strain WVU-1853, or M. iowae strain I-695 at concentration of 256 ng.

Animals↗

PubMed1990

Targeted cytogenetic analysis of gastric tumors by in situ hybridization with a set of chromosome-specific DNA probes.

Fluorescent in situ hybridization (FISH) with biotinated chromosome-specific repetitive DNA probes was used for the cytogenetic study of ten gastric adenocarcinomas. All tumors (eight male, two female patients) were histologically moderately or poorly differentiated and nine of ten had metastasized to regional lymph nodes. The authors applied a set of satellite DNA probes, specific for chromosomes 1, 7, 17, X, and Y in order to detect numerical chromosome aberrations in freshly isolated tumor cell nuclei. Normal diploid human lymphocyte nuclei and, in a number of cases, normal gastric mucosa served as controls. Parallel with the hybridization experiments DNA flow cytometric study of acridine orange (AO)-stained tumor cells was carried out. By means of FISH the authors found seven cases to be aneuploid, the other three cases appeared diploid. This was confirmed by DNA flow cytometric analysis with AO. Furthermore, loss of the Y chromosome in a high percentage of cells was seen by FISH in six of eight tumors from male patients. In the other two male samples a possible loss was observed in a small proportion of cells (15%). In three patients from whom the authors had normal gastric mucosa the Y loss was restricted to the tumor cells. These data indicate that in situ hybridization with chromosome-specific repetitive DNA probes can serve as a cytogenetic tool for the analysis of interphase nuclei of solid human tumors, at least with respect to the detection of numerical chromosome abnormalities.

Adenocarcinoma↗

PubMed1993

[Design of a chromosomal DNA probe species specific for Yersinia pestis].

A 14.8 kb DNA fragment from the chromosome of Yersinia pestis TWJ was cloned and the restriction map constructed. The fragment designated as T16 and its subfragments were tested in dot-hybridization with strains of Yersinia genus and other members of Enterobacteriaceae. A species-specific DNA probe (designated MK) was constructed on the basis of the T16 fragment. As judged from restriction analysis, blot-hybridization experiments and, partially, sequencing, significant homology exists between the MK DNA probe and this one developed by Bardarov et. al. (1990). A repeated sequence in two copies was discovered in the MK fragment.

Base Sequence↗

PubMed1989

Detection of Plasmodium falciparum infection in human patients: a comparison of the DNA probe method to microscopic diagnosis.

We have previously reported the isolation and testing of a DNA probe specific for the detection of Plasmodium falciparum. Field studies to compare the sensitivity and specificity of the DNA probe with that of light microscopy have been performed. In 2 studies in Thailand, 1,397 patients were tested. Microscope slides were prepared in a standard fashion and examined by clinical technicians and expert microscopists. The DNA probe method compares favorably in sensitivity with routine microscopy, detecting parasite densities as low as 40 parasites/microliters blood in the first study and, after modifications, 20-25 parasites/microliters blood in the second. Modifications included the elimination of salt from the lysis buffer, increasing the pH of the lysis buffer, and use of nylon based hybridization membranes instead of nitrocellulose. The DNA probe method offers the advantage of a standardized procedure that can be used in a batchwise fashion on a large number of samples.

Adolescent↗

PubMed1992

Evaluation of a DNA probe to identify enteroaggregative Escherichia coli from children with diarrhoea in Bangladesh.

Six hundred and seventy-five Escherichia coli isolates obtained from 225 diarrhoeal children less than five years of age were tested for adherence to HeLa cells and for hybridisation with DNA probes for genes conferring aggregative adherence (AggA), localised adherence (LA) and diffuse adherence (DA) to assess the usefulness of a recently developed DNA probe for AggA of enteroaggregative E. coli (EAggEC). The strains were further analysed with the DNA probes for heat--labile enterotoxin (LT), heat--stable enterotoxin (ST), Shiga--like toxins (SLT I and SLT II) and for enteroinvasiveness and adherent strains were all negative for these properties. The HeLa cell assay and DNA probe assays showed excellent agreement in identifying LA and DA positive isolates. However, significant disparities occurred in the case of AggA positive isolates, and the DNA probe failed to identify 31.9% (15 of 47) of the EAggEC identified by the HeLa cell adherence assay. The failure of the DNA probe to identify all the EAggEC indicated that there may be a high degree of genetic heterogeneity for the expression of AggA, and development of more DNA probes is necessary to detect all the possible genetic variants of EAggEC.

Bacterial Adhesion↗

PubMed1995

DNA probes and PCR for diagnosis of parasitic infections.

DNA probe and PCR-based assays to identify and detect parasites are technically complex; however, they have high sensitivity, directly detect parasites independent of the immunocompetence or previous clinical history of the patient, and can distinguish between organisms that are morphologically similar. Diagnosis of parasites is often based on direct detection by microscopy, which is insensitive and laborious and can lack specificity. Most PCR-based assays were more sensitive than DNA probe assays. The development of PCR-based diagnostic assays requires multiple steps following the initial selection of oligonucleotide primers and reporter probe. Generally, the ability to detect the DNA of one parasite was attained by PCR; however, advances in the preparation of samples for PCR (extraction of DNA while removing PCR inhibitors) will be required to achieve that sensitivity with human specimens. Preliminary PCR systems have been developed for many different parasites, yet few have been evaluated with a large number of clinical specimens and/or under field conditions. Those evaluations are essential for determination of clinical and field utility and performance and of the most appropriate application of the assay. Several situations in which PCR-based diagnosis will result in epidemiologic, medical, or public health advances have been identified.

Animals↗

PubMed1992

DNA probes for detecting Coxiella burnetii strains.

Methods have been developed for the rapid detection of C. burnetii by specific hybridization of labelled DNA probes to rickettsial plasmid DNA sequences present in clinical samples. One DNA probe detects all C. burnetii strains, while additional probes differentiate, between organisms associated with chronic or acute disease. Using these probes, C. burnetii can be identified in blood, urine, and tissue samples. The plasmid-derived DNA probes detect as few as 10(4) organisms and less than 1 ng of Coxiella DNA. Host-cell DNA has no effect on the hybridization signal from C. burnetii DNA, and these probes do not cross-react with a variety of microorganisms, including both common laboratory contaminants and organisms that cause clinical symptoms similar to those of Q fever. The sensitivity of the assay is markedly enhanced when the procedure employs the polymerase chain reaction (PCR) to amplify C. burnetii DNA. This requires construction of oligonucleotide primers to DNA sequences flanking the target region of the DNA being amplified. For C. burnetii detection, several sets of primers have been prepared. One set is derived from the QpH1 H fragment, a region that is shared by all C. burnetii plasmids (homologous sequences are also present in the plasmidless strains of C. burnetii). The H primers detect all strains of C. burnetii. To differentiate between C. burnetii strains, additional primers, specific for DNA sequences that are unique either to chronic or acute disease-related strains of C. burnetii are employed. PCR amplifies target sequences up to 10(6)-fold. When DNA hybridization is used in conjunction with PCR, the test can detect less than 10 C. burnetii cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute Disease↗