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PubMed1992

[Detection of Campylobacter species by using polymerase chain reaction and nonradioactive DNA probes. II. PCR direct sequencing of the Campylobacter DNA].

We have developed a sensitive DNA hybridization assay for the detection and identification of Campylobacter species which are recognized as important pathogens of acute diarrheal disease in humans. This technique utilizes DNA probes complementary to nucleotide sequences present in 16S ribosomal RNA (rRNA) of C. jejuni, C. coli, C. laridis, C. fetus, C. fetus subsp. fetus, C. fetus subsp. venerealis and C. hyointestinalis, and polymerase chain reaction. The partial sequence of DNAs encoding the Campylobacter rRNA was first analyzed by direct solid phase sequencing in order to select suitable DNA probes. Amplified target DNA of 240 base pairs could be resolved on ethidium bromide-stained gels, and hybridized with DNA probes conjugated to alkaline phosphatase. In identification experiments, one of the 10 probes tested here gave a positive hybridization reaction with C. jejuni, C. coli and C. hyointestinalis but not with other Campylobacter species. The other was specifically reactive with C. fetus, C. fetus subsp. fetus and C. fetus subsp. venerealis. When applied to stool specimens, a good correlation was found between the results obtained by the present assay and by biochemical tests. These findings suggest that the nonradioactive probe assay can be used as the practical criterion for differentiating Campylobacter species.

Base Sequence↗
PubMed1989

Nonradioactive, photobiotin-labelled DNA probes for routine diagnosis of viroids in plant extracts.

Avocado sunblotch viroid (ASBV), coconut cadang cadang viroid (CCCV), chrysanthemum stunt viroid (CSV) and potato spindle tuber viroid (PSTV) were detected in plant extracts by dot-blot hybridization using nonradioactive photobiotin-labelled nucleic acid probes. Recombinant DNA probes, containing full-length monomer viroid inserts in the plasmid vectors pSP64 or pUC9, were biotinylated with photobiotin and used as sonicated double-stranded DNA fragments. Using fresh leaf material, a general method (suitably modified for avocado tissue) was developed for the rapid preparation of purified nucleic acid extracts. Plant extracts from a range of field samples were spotted onto nitrocellulose, subjected to hybridization and the biotin-labelled DNA bound to the target nucleic acid was detected with an avidin-alkaline phosphatase conjugate. Under the stated hybridization and washing conditions, each individual viroid probe was specific. Each viroid was readily detected with a sensitivity similar to that obtained with the same (or a like) probe labelled with 32P. Healthy plant extracts gave colourless spots.

Affinity Labels↗
PubMed1990

Development of a recombinant Anaplasma marginale DNA probe.

An Anaplasma marginale DNA probe has been developed by using an improved method for the isolation of genomic DNA. Purified genomic A. marginale DNA from the St. Croix isolate was partially digested with Sau 3A1 into fragments (greater than or equal to 5.0 kb). The restriction fragments were cloned using standard techniques in the pBR322 vector and used to transform E. coli (DH5) host cells. The recombinant A. marginale DNA library was screened by the colony lifting procedure. Colonies containing plasmids with A. marginale DNA inserts were identified by hybridization with a genomic A. marginale DNA radiolabeled probe (32P). Seven recombinant A. marginale DNA probes were evaluated by dot-blot in vitro hybridization assays to identify candidates as diagnostic tools in bovine anaplasmosis studies. Specificity and sensitivity experiments were carried out by using heterologous and homologous DNAs. The heterologous panel contained bovine DNA (WBC) and blood parasites DNA from Babesia bovis (Bb), Babesia bigemina (Bbi), Eperythrozoon suis (Es) and Eperythrozoon wenyoni (Ew). The homologous DNA panel included A. marginale DNAs of 12 different isolates which were isolated in the Caribbean, Mexico, and the U.S.A. The selected diagnostic probe was identified as pSt. Croix A1, and labeled with 32P by using in vitro nick translation and random primer techniques. The pSt. Croix A1 probe demonstrated 100% specificity and high sensitivity by hybridization in dot blotting and Southern blotting. The probe can detect 500-1000 infected erythrocytes per microliters which corresponds to a parasitemia of less than 0.01%. The A. marginale DNA insert was approximately 6.4 kb in size and a partial restriction map has been constructed.

Anaplasma↗
PubMed1996

Analysis of the DNA content distribution of micronuclei using flow sorting and fluorescent in situ hybridization with a centromeric DNA probe.

The DNA content distributions of micronuclei induced in mouse 3T3 cells by ionizing radiation and chemicals was measured by flow cytometry. For a quantitative understanding of these distributions, micronuclei with increasing DNA contents were sorted and analysed for the presence of centromeric signals using fluorescent in situ hybridization (FISH) with a mouse centromeric gamma satellite probe. Radiation-induced micronuclei were found to be produced mainly by chromosome fragments, whereas micronuclei induced by the tear gas chlorobenzylidene malonitrile (CS) were found to be produced mainly by whole chromatids. In contrast, micronuclei induced by vinblastine (VBL) were, according to the shape of their DNA content distributions, produced mainly by whole chromosomes and by combinations of two or more whole chromosomes. With increasing DNA content, micronuclei induced by ionizing radiation also contained one or more whole chromosomes, whereas micronuclei induced by CS or VBL were found to contain several whole chromatids or chromosomes respectively. Computerized random breakage of chromosomes and random combination of chromosome fragments, whole chromatids and whole chromosomes were used according to the FISH results to simulate the measured DNA content distributions of micronuclei. A good agreement was obtained between measured and simulated distributions of micronuclei as well as between results of the measured frequency of micronuclei showing centromeric signals as a function of their DNA content and those predicted by the simulations. These results demonstrate the usefulness of flow cytometry and sorting combined with the FISH technique and computer simulations for producing a more detailed analysis of mechanisms of micronucleus induction.

3T3 Cells↗
PubMed1991

Tetracycline fiber therapy monitored by DNA probe and cultural methods.

Oligonucleotide DNA probe and selective cultural methods were compared in their ability to monitor 6 putative periodontal pathogens in a study evaluating local tetracycline fiber therapy. Subgingival plaque was sampled from 4 sites in each of 20 subjects. Samples were taken before and after therapy from sites assigned to the following test groups: tetracycline (TC) fiber, scaling and root planing, control fiber, and untreated. Each sample was analyzed by both DNA probe and cultural methods. Total anaerobic cultivable counts, Porphyromonas (Bacteroides) gingivalis and Prevotella intermedia (Bacteroides intermedius) were enumerated on nonselective blood agar. Actinobacillus actinomycetemcomitans, Eikenella corrodens, Fusobacterium nucleatum and Wolinella recta were isolated on selective media. TC fiber therapy and scaling reduced total cultivable counts from an initial value of 1 x 10(7) to approximately 2 x 10(5) following therapy. Total counts at untreated sites and at sites with control fibers did not change from baseline. A. actinomycetemcomitans and E. corrodens were detected more frequently by the cultural method; the other monitored species were detected more frequently by DNA probes than by the cultural methods. Agreements between methods were: 77.2% for A. actinomycetemcomitans; 72.2% for P. intermedia; 75.6% for E. corrodens; 39.4% for F. nucleatum; 35.6% for P. gingivalis; and 68.9% for W. recta. Limitations of the selective cultural methods used probably contributed to the discrepancies for P. gingivalis and F. nucleatum. DNA probe and cultural methods indicated comparable levels of suppression of the monitored species following TC fiber therapy and scaling. The microbiota of control fiber and untreated sites did not appear to be significantly altered by either method.

Actinobacillus↗
PubMed1985

Comparison of radio-labeled DNA probe with a nonisotopic probe for assay of serum hepatitis B virus DNA.

A biotin-labeled DNA probe was compared to a 32P radio-labeled DNA probe for the detection of serum hepatitis B virus (HBV) DNA. Serum specimens were treated with proteolytic enzyme and detergent. DNA was extracted using phenol, denatured in sodium hydroxide and applied to a nitrocellulose filter paper using a vacuum filter device. The nitrocellulose filters were then incubated with either the biotin-labeled or the radio-labeled probe. Annealing of the probe, indicating the presence of HBV-DNA in the sample, was detected either by autoradiography for the 32P-labeled probe or by measuring the presence of an acid phosphatase attached to a streptavidin molecule for the biotin-labeled probe. Using the same 2-day time to complete the assays, excellent correlation of the qualitative and semiquantitative measurements were obtained using 20 HBsAg-positive and 9 HBsAg-negative sera. The nonisotopic assay detected 1.0 pg of HBV-DNA, a sensitivity comparable to reported sensitivities of 32P-labeled HBV-DNA probes when similar assay times are used. 0.02 pg/microliter of HBV-DNA was detected in a normal serum to which HBV-DNA in a recombinant plasmid was added. Our results indicate that the biotin-labeled HBV-DNA probe is approximately as sensitive as the radio-labeled probe for the detection of HBV-DNA using a similar assay time. Isotopic probe assays are more sensitive with longer assay times. The biotin-labeled probe offers the advantage of a longer shelf life and a nonisotopic assay procedure.

Acid Phosphatase↗
PubMed1991

[A comparative study of pulmonary disease due to Mycobacterium avium and M. intracellulare identified by a newly developed DNA probe (Gen-Probe].

By using DNA Probe, cases due to M. avium complex in Japan were grouped into those M. avium and M. intracellulare and their clinical patients were compared by multifactorial analysis. A total of 179 cases was studied (103 due to M. avium and 76 due to M. intracellulare). M. avium cases were found more commonly around Tokyo and north, whereas M. intracellulare cases were seen more frequently in the western part of Japan. Background factors (sex, age, past history, complications, bacterial status, mode of detection, and symptoms) were similar among the cases due to M. avium and M. intracellulare. No difference was seen in the chest X-ray. Weighing up to the disappearance of bacterial excretion revealed heavier in the quantity of bacterial excretion in M. avium cases, but not much in chemotherapy. In M. intracellulare cases on the other hand, chemotherapy played a more weighed role. In both groups, EB was weighed heavier among the drugs. Among the additionally used drugs, SM was useful in M. avium and KM in M. intracellulare. RFP was useful in combined therapy. The usefulness of INH was lower. In areas western to Osaka and vicinity, MAC was reported to exert favorable effect. In case this is true, higher prevalence of M. intracellulare and the use of KM, EB, RFP in these areas may explain such phenomena.

Adult↗
PubMed1990

Echinococcus multilocularis: characterization of a DNA probe.

A 0.6 kb DNA fragment has been isolated from a genomic sublibrary of the cestode Echinococcus multilocularis. This DNA-fragment showed a strong hybridization signal to 32PdCTP labeled total DNA prepared from E. multilocularis metacestode material. The fragment was subcloned into the Escherichia coli vector Bluescript BS+ resulting in the recombinant plasmid pAL1. The recombinant parasite DNA probe was labeled by biotinylation and hybridized to Southern blots of resolved EcoRI/PstI digested genomic DNA originating from E. multilocularis, E. granulosus and other helminth species (Taenia hydatigena, T. crassiceps, T. saginata, Mesocestoides corti, Hymenolepis diminuta, Moniezia expansa). The Southern blot hybridization experiments revealed that the DNA probe pAL1 was specific for E. multilocularis and E. granulosus. By comparison of the hybridization banding patterns a clear discrimination between E. multilocularis and E. granulosus was possible at the genome level. Furthermore, pAL1 was used to detect genetic variation within a set of different E. multilocularis isolates which had been experimentally maintained in mice by parasite tissue transplantation.

Animals↗
PubMed1999

Strand displacement amplification and homogeneous real-time detection incorporated in a second-generation DNA probe system, BDProbeTecET.

BACKGROUND: Amplified DNA probes provide powerful tools for the detection of infectious diseases, cancer, and genetic diseases. Commercially available amplification systems suffer from low throughput and require decontamination schemes, significant hands-on time, and specially trained laboratory staff. Our objective was to develop a DNA probe system to overcome these limitations. METHODS: We developed a DNA probe system, the BDProbeTecTMET, based on simultaneous strand displacement amplification and real-time fluorescence detection. The system uses sealed microwells to minimize the release of amplicons to the environment. To avoid the need for specially trained labor, the system uses a simple workflow with predispensed reagent devices; a programmable, expandable-spacing pipettor; and the 96-microwell format. Amplification and detection time was 1 h, with potential throughput up to 564 patient results per shift. We tested 122 total patient specimens obtained from a family practice clinic with the BD ProbeTecET and the Abbott LCx(R) amplified system for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae. RESULTS: Based on reportable results, the BDProbeTecET results for both organisms were 100% sensitive and 100% specific relative to the LCx. CONCLUSIONS: The BDProbeTecET is an easy-to-use, high-throughput, closed amplification system for the detection of nucleic acid from C. trachomatis and N. gonorrhoeae and other organisms.

Chlamydia trachomatis↗
PubMed1991

Producing single-stranded DNA probes with the Taq DNA polymerase: a high yield protocol.

We report an efficient procedure to synthesize either single- or double-stranded probes labeled with biotin-11-dUTP, biotin-21-dUTP or digoxigenin-11-dUTP. To produce the single-stranded probe, only a single primer is utilized in a Taq polymerase amplification of 55 cycles. A cytomegalovirus probe is presented. This procedure allows easy production of nonradioactively labeled pure single-stranded probes of any desired length and specificity.

Base Sequence↗
PubMedDate not supplied

Detection of Mycoplasma gallisepticum infection in field samples using a species-specific DNA probe.

Species-specific DNA probes for Mycoplasma gallisepticum (MG) were compared with serologic and isolation procedures as a routine diagnostic tool on field specimens acquired from chicken flocks experiencing egg-production losses and suspected of MG infection. The MG DNA probe clearly identified MG directly from tracheal specimens within 2 days, unlike the 7 to 10 days required for culture procedures. Cross-reaction of MG with M. synoviae continues to be a stumbling block in the serum plate agglutination test.

Agglutination Tests↗
PubMed1990

Investigation of paternity establishing without the putative father using hypervariable DNA probes.

Seven kinds of DNA probes which recognize hypervariable loci were applied for paternity test. The putative father was decreased and unavailable for the test. The two legitimate children and their mother (the deceased's wife) and the four illegitimate children and their mother (the deceased's kept mistress) were available for analysis. Paternity index of four illegitimate child was investigated. Allelic frequencies and their confidence intervals among unrelated Japanese individuals were previously reported from our laboratory, and co-dominant segregation of the polymorphism was confirmed in family studies. Cumulative paternity indices of four illegitimate children from 16 kinds of standard blood group markers were 165, 42, 0.09, and 36, respectively. On the other hand, cumulative paternity indices from 7 kinds of DNA probes are 2,363, 4,685, 57,678, and 54,994, respectively, which are 14, 113, 640, 864, and 1,509 times higher than that from standard blood group markers. The DNA analyses gave nearly conclusive evidence that the putative father was the biological father of the children. Especially, the paternity relation of the third illegitimate child could not be established without the DNA analyses. Accordingly, DNA polymorphism is considered to be informative enough for paternity test.

DNA↗
PubMed1991

Direct in-gel hybridization, without blotting, using nick-translated cloned DNA probe.

Hybridization of DNA and RNA to labeled probes is central to molecular biology. A method is described here for hybridization of cloned DNA probes directly to DNA in agarose gels. This in-gel hybridization method has several advantages over conventional techniques using transfer to membranes. It is extremely rapid, highly sensitive, less expensive and particularly suited for high molecular weight genomic DNA analysis.

Cloning, Molecular↗
PubMed1993

In situ hybridization at the electron microscopic level using a bromodeoxyuridine labeled DNA probe.

A bromodeoxyuridine (BrdU) labeled DNA probe was used for in situ hybridization at the electron microscopic (EM) level. A BrdU labeled DNA probe was hybridized in situ to cryostat sections of paraformaldehyde fixed OCT compound embedded cultured HL-60 cells. After hybridization, some sections were incubated with FITC-conjugated anti-BrdU monoclonal antibody for fluorescence microscopy (FM), and others were embedded in Quetol for electron microscopy (EM). The ultrathin sections of Quetol-embedded specimens were incubated with the anti-BrdU monoclonal antibody and the immunoglobulin: gold colloid. In both FM and EM studies, the signals were concentrated in the rough endoplasmic reticulum. Moreover, some label was arranged from the nucleus to the cytoplasm at the EM level. Relatively simple methods using the BrdU labeled DNA probe for the detection of the defined nucleic acid sequence with reasonable tissue preservation and high resolution are described here. This method may be useful for developmental and disease related studies of specific mRNA in cells and tissues.

Antibodies, Monoclonal↗
PubMed1987

A solution hybridization assay for ribosomal RNA from bacteria using biotinylated DNA probes and enzyme-labeled antibody to DNA:RNA.

Rapid, convenient and non-isotopic nucleic-acid hybridization methods are needed for this technology to have practical use in clinical diagnostic tests. A method for hybridization of RNA with a DNA probe in solution followed by capture and measurement of the hybrid is described. DNA probes complementary to 23S rRNAs from Escherichia coli and Bacillus subtilis were labeled with a photoactivable biotin reagent. Hybridization of the biotinylated probes with rRNA was complete in less than 5 min. The resultant hybrids were allowed to bind simultaneously to succinylated avidin immobilized on latex and to beta-galactosidase-labeled Fab' fragments of a monoclonal antibody-specific for DNA:RNA. Finally, beta-galactosidase associated with the captured hybrids was measured colorimetrically. The hybridization method can detect less than 1000 bacteria per assay and has broad specificity to permit detection of the various genera of bacteria that infect the urinary tract.

Animals↗
PubMed1990

[DNA hybridization and identification of Leptospira serogroup icterohaemorrhagiae with Leptospira recombinant DNA probes].

Two recombinant DNA probes-PLIpso1 (15kb) and PLIEc34 (4kb), derived from Leptospira serovaricterohaemorrhagiae genomic libraries, were applied for the hybridization and identification of 13 strains of Leptospira in serogroup icterohaemorrhagiae. Difference in hybridization signal in combination with the banding pattern provide a good way for identification of serovars and strains. The recombinant DNA, specific to L. serogroup icterohaemorrhagiae, hybridized with a limited number of DNA fragments which had been digested by several restriction endonucleases. The less complex banding pattern and higher sensitivity facilitate characterization of various serovars and strains in serogroup icterohaemorrhagiae. In general the DNA patterns recognized by both probes have extensive genomic homology in same serovar (but still can distinguish strains in same serovar by some unique bands) and apparent difference in various serovars (especially serovars naam, nanxi and honghe). The results indicated that Southern blotting with recombinant DNA probe might provide tools for identification, characterization and analysis of leptospira.

Blotting, Southern↗
PubMed1987

Rapid identification of Mycobacterium avium complex in culture using DNA probes.

A commercial DNA probe procedure for identification of Mycobacterium avium complex isolates was evaluated for accuracy and applicability for use in the clinical laboratory. The test (Gen-Probe Rapid Diagnostic System for Mycobacterium Avium Complex; Gen-Probe Corp., San Diego, Calif.) uses hybridization in solution of two 125I-labeled cDNA probes. One probe is complementary to rRNA from M. avium, and the other is complementary to rRNA from M. intracellulare. Results are expressed as absolute percent hybridization, with values greater than or equal to 10% considered positive. The procedure accurately identified all 134 M. avium complex isolates and concomitantly identified them to species level. There were no false-positives with 66 other mycobacteria, including 22 M. tuberculosis and 18 M. kansasii isolates, or with 8 Nocardia isolates. The mean percent hybridization (+/- the standard deviation) of M. avium probe-positive isolates (94 isolates) was 48.0 +/- 9.9 (range, 11.5 to 72.7); for M. intracellulare (40 isolates), it was 45.7 +/- 8.8 (range, 22.7 to 60.7). Among the 74 non-M. avium complex isolates, the percent hybridization range was 1.0 to 4.2, except for a single value of 9.7 which was less than 5 when the test was repeated. Four M. avium complex isolates reacted positively with both probes on initial testing, and three were confirmed. On repeat testing of subcultures, each reacted with only one probe, suggesting the presence of a mixed culture. The procedure can be completed in as little as 2 h and could easily be performed in most clinical laboratories.

Culture Media↗