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PubMed1991

Detection of the human immunodeficiency virus genome with a biotinylated DNA probe generated by polymerase chain reaction.

A non-radiolabelled DNA probe was developed for detection of the human immunodeficiency virus type 1 (HIV-1) genome using the polymerase chain reaction (PCR) technology. Primers amplifying a 395 base pair segment of a portion of the polymerase region of the HIV-1 genome were used both to amplify sample target DNA and to generate a biotinylated DNA probe used in Southern blot hybridization. This probe performed as well as one produced by nick translation using biotinylated nucleotides or an enzyme labelled oligonucleotide probe.

Bacterial Proteins↗

PubMed1998

A DNA probe for identification of larvae of the commercial surfclam (Spisula solidissima).

A molecular DNA probe was developed for identification of larvae of the commercial surfclam Spisula solidissima (family Mactridae), to distinguish early-stage veligers from larvae of other common bivalve species in a study of surfclam settlement and recruitment on the New Jersey continental shelf. For discrimination of S. solidissima from other common bivalve species at the study site (almost all of which belong to different families), an oligonucleotide designed from the nucleotide sequence of the 18S ribosomal RNA gene provides a useful and sensitive family-specific probe and primer. For discrimination between S. solidissima and Mulinia lateralis (both members of Mactridae), the 18S rRNA gene was examined by restriction fragment length polymorphism (RFLP) analysis. A combination of the DNA probe and RFLP analysis provides a positive identification of S. solidissima and M. lateralis larvae in coastal plankton samples.

Animals↗

PubMed1995

Construction and characterization of a DNA probe for distinguishing strains of Aspergillus flavus.

Repetitive DNA sequences have proven useful and reliable characters in evaluating genetic relatedness of strains at different levels of taxonomic classification. A DNA probe was constructed to distinguish among strains of Aspergillus flavus by DNA fingerprinting techniques. Chromosomal DNA of A. flavus var. flavus NRRL 6541 was partially digested with EcoRI and ligated to a Lambda Dash bacteriophage vector. Four lambda clones were identified which displayed multiple and distinct bands when hybridized with chromosomal DNA from seven strains of A. flavus var. flavus digested with either EcoRI or PstI. One of these clones was chosen for further analysis and was subcloned into pUC19. The subclone, pAF28, contained a 6.2-kb chromosomal DNA insert and was able to distinguish among strains characterized by K. E. Papa (Mycologia 78:98-101, 1986) as belonging to 22 different vegetative compatibility groups. The subclone identified unique banding patterns when hybridized to genomic DNA digested with PstI. The cloned probe may be species specific as it hybridized with the DNA of all isolates of A. flavus tested in addition to strains recognized as varieties of A. flavus (e.g., A. flavus var. oryzae, A. flavus var. parasiticus, and A. flavus var. sojae). pAF28 hybridized to a single band on a Southern blot with Aspergillus nomius DNA but did not hybridize with the DNA of other fungal species tested including Aspergillus ochraceus, Aspergillus auricomus, Aspergillus alliaceus, Fusarium moniliforme, and Penicillium thomii.

Aspergillus flavus↗

PubMed1991

Identification of thermotolerant Campylobacter species from poultry using an enzyme-labelled oligonucleotide DNA probe.

A commercially available enzyme-labelled DNA probe for human Campylobacter strains has been tested and also found to hybridize with DNA from C. jejuni and C. coli isolates from poultry. DNA from 11 enteric, non-campylobacter organisms, included in the test as negative controls, failed to hybridize with the probe indicating that the probe might be used for identification of campylobacter from poultry too.

Animals↗

PubMed1989

A biotinylated DNA probe to detect bacterial cells in artificially contaminated foodstuffs.

A biotin-labelled DNA probe was used in a dot-blot hybridization test to demonstrate the presence of Escherichia coli in a variety of artificially contaminated foodstuffs. Positive hybridization was detected by using a streptavidine/polyalkaline phosphatase conjugate to generate an insoluble coloured precipitate in the presence of an appropriate dye. The colour intensity was measured with a computer-controlled image analysis system which assessed objectively the hybridization signal produced by each sample. The method was capable of distinguishing positive hybridization at cell concentrations exceeding 10(4) cells/dot-blot, equivalent to 2 x 10(7) cells/g food, and had none of the drawbacks normally associated with the use of radioactively labelled DNA in hybridization techniques. The procedure is highly specific and takes less than 30 h. Many samples can be screened simultaneously and the procedure can be used to detect any species for which a suitable DNA probe is available.

Bacteria↗

PubMed1991

Isolation and characterization of a species-specific DNA probe for Candida albicans.

As a model for the isolation of species-specific sequences of DNA, we isolated and characterized a species-specific DNA fragment from the Candida albicans genome. We used a series of differential colony hybridization experiments, in which a C. albicans genomic library was hybridized with genomic DNA probes from related organisms to minimize the number of potentially specific C. albicans DNA fragments to be tested. Six clones were tested by dot blot analysis, and one of them, a 1348 bp EcoRI DNA fragment, was confirmed as specific for C. albicans. This species-specific fragment could be utilized as a DNA probe for rapid, sensitive, and specific diagnosis of C. albicans DNA in clinical specimens.

Base Sequence↗

PubMed2004

Use of a high-density DNA probe array for detecting mutations involved in rifampicin resistance in Mycobacterium tuberculosis.

A Mycobacterium high-density DNA probe array designed to detect rpoB mutations conferring rifampicin resistance in Mycobacterium tuberculosis was evaluated. The rpoB hybridisation patterns produced by 41 susceptible (RifS) and 59 rifampicin-resistant (RifR) clinical isolates of M. tuberculosis were compared with the results of conventional dideoxynucleotide sequencing of the rpoB gene. For all the RifR isolates, the rpoB hybridisation patterns correlated with the rpoB sequencing results. Among the 59 isolates, 11 distinct amino-acid changes were detected by the DNA probe array. Of these, 36 (61%) corresponded to replacement of the serine residue found in position 531 (S531L in 34 isolates and S531W in two isolates), 16 (27%) affected histidine 526 (five H526D, five H526Y, four H526L, one H526N and one H526R), four (6.8%) replaced aspartate 516 with a valine, and one (1.7%) replaced glutamine 513 with a leucine. Deletion of the asparagine residue at position 519 was detected in one isolate susceptible to rifampicin, but yielding c. 0.1% resistant colonies on rifampicin-containing medium. No mutation was detected in the rpoB region from one isolate yielding c. 5% of resistant colonies on rifampicin-containing medium. Finally, a D516Y substitution was detected in association with an unexpected mutation, G523W, not tiled on the DNA probe array, but which could be detected by analysing the hybridisation pattern obtained with the wild-type probes covering codon 523. In conclusion, the Mycobacterium probe array is a promising approach to rapid detection of mutations involved in rifampicin resistance in M. tuberculosis.

Amino Acid Substitution↗

PubMed1995

Development of a specific biotinylated DNA probe for the detection of Renibacterium salmoninarum.

A specific DNA probe for the identification of Renibacterium salmoninarum, the causative agent of bacterial kidney disease (BKD), was developed from one of 3 clones pRS47, pRS49, and pRS26 of 5.1 kb, 5.3 kb, and 11.3 kb, respectively. The biotinylated pRS47/BamHI insert probe was tested on 3 dilutions of DNA extracted from 3 strains of R. salmoninarum and from 1 strain each of Arthrobacter protophormiae, Aeromonas salmonicida, Corynebacterium aquaticum, Carnobacterium piscicola, Listonella anguillarum, Micrococcus luteus, Pseudomonas fluorescens, Vibrio ordalii, and Yersinia ruckeri. In a dot blot assay, this probe hybridized only with the DNA from the R. salmoninarum strains. When used on kidney samples from fish challenged with R. salmoninarum, the dot blot hybridization assay with the probe was found to be as sensitive as culture. In a fluorescent antibody test, samples that were negative in culture and dot blot hybridization showed no more than one fluorescing cell in 50 microscopic fields examined. This DNA probe, therefore, has the potential for use in the diagnosis of BKD of fish.

Actinomycetales↗

PubMed1992

Evaluation of a chemiluminescent DNA probe assay for the rapid confirmation of Listeria monocytogenes.

A Listeria monocytogenes-specific, acridinium-ester-labelled DNA probe was evaluated in a chemiluminescent homogeneous protection assay (HPA) for the rapid confirmation of suspect L. monocytogenes colonies from blood agar plates. The HPA uses an acridinium-ester-labelled chemiluminescent DNA probe in a free-solution hybridization format. After the DNA probe hybridized with the target ribosomal RNA, the acridinium label on the unhybridized probe was inactivated by a chemical differential hydrolysis step. Formation of a hybrid between probe and target was detected in a luminometer after the addition of a detection reagent. The assay can be completed in 30 to 45 min and allows for simultaneous processing of several (50-100) samples. The probe showed 100% sensitivity and 100% specificity for L. monocytogenes when evaluated in the HPA against L. monocytogenes, other Listeria species and other Gram-positive bacteria. The lower detection limit of the HPA was between 10(4) and 10(5) cells. In an evaluation with 296 bacterial colonies isolated from food, the HPA colony confirmation showed 100% agreement with conventional biochemical characterization. HPA will be useful for the rapid confirmation of L. monocytogenes isolated from food and clinical specimens.

Acridines↗

PubMed1993

[Genetic distances between various ethnic populations calculated on the basis of polymorphism of DNA detected by the hypervariable phage M13 DNA probe].

The frequencies of different electrophoretic bands in DNA "fingerprint" detected by the phage M13 DNA probe in six Russian populations from the Kirov district and Krasnodar, one from Chuvashia and one from Tuva were compared with each other and pseudo-genetic distances by Nei were calculated. The results corresponded well with a presumable extent of similarity between the populations observed.

Bacteriophage M13↗

PubMed1988

The use of whole-cell DNA probes for the identification of Bacteroides intermedius isolates in a dot blot assay.

Bacteroides intermedius includes two distinct groups of organisms that are phenotypically indistinguishable by conventional methods. These two groups are represented by the type strain of the species ATCC 25611T (B. intermedius type I) and by ATCC 33563 (B. intermedius type II). Members of each group can be distinguished from each other by analysis of the cellular protein composition by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and by DNA-DNA homology studies, because they share less than 40% homology. The purpose of this study was to prepare specific DNA probes for the two groups of Bacteroides intermedius and to test them against field isolates. Whole-cell DNA probes were prepared from B. intermedius types I and II and tested against 253 field strains of Bacteroides which had been identified by conventional phenotypic tests as B. intermedius. Of these, 170 (67%) hybridized with the B. intermedius type I DNA probe, 28 (11%) with the type II, and 23 (9%) failed to react with the B. intermedius probes but did hybridize with either B. melaninogenicus, B. loescheii, or B. corporis whole-cell DNA probes. The 32 (13%) remaining isolates failed to hybridize with any of the five Bacteroides probes or with probes to B. asaccharolyticus, B. buccae, B. buccalis, B. denticola, B. gingivalis, B. oralis, or B. oris. These data demonstrate the usefulness of whole-cell DNA probes for the identification of phenotypically similar or identical field isolates.

Bacteroides↗

PubMed1991

Capsular typing of Haemophilus influenzae with a DNA probe.

We describe the use of a DNA probe for genotyping clinical isolates of Haemophilus influenzae. The probe, containing capsulation genes, differentiates between the six Haemophilus serotypes in a Southern blotting procedure. It also hybridizes with a distinctive pattern to DNA from capsule-deficient mutants of serotype b strains, while failing to hybridize to DNA from typical clinical isolates of non-serotypable H. influenzae. The probe can thus resolve issues of serotyping uncertainty such as arise, for example, when capsulate strains are found to have lost reactivity with serotyping reagents after storage or transmission from one laboratory to another. The probe has proved useful in the evaluation of Haemophilus infections in infants following administration of H. influenzae type b vaccine. In an illustrative example, the probe was used to resolve serotyping ambiguity in a case of Haemophilus bacteraemia in a vaccine recipient, providing compelling evidence that the organism responsible was neither type b nor derived from a type b strain. The widespread introduction of vaccines against H. influenzae type b disease will increase the importance of the precise identification of strains infecting immunized children. This need can only be met by the development of 'gold standards' such as capsulation gene probes.

Animals↗

PubMedDate not supplied

Identification of Neisseria gonorrhoeae with the ORTHOProbe DNA probe test.

We evaluated and compared the ORTHOProbe DNA hybridization test (Ortho Diagnostic Systems, Inc., Raritan, NJ) with conventional carbohydrate fermentation and commercial rapid tests for identifying gonococci (GC). The ORTHOProbe system employs a biotinylated DNA probe that hybridizes specifically with chromosomal target sequences of GC. Hybridization is detectable visually following the addition of a streptavidin-peroxidase conjugate in a rapid 10-min. assay. A total of 200 clinical isolates resembling Neisseria (gram-negative, oxidase-positive diplococci) cultured from various sites were tested. The ORTHOProbe test reacted strongly with all GC (94) and not with nongonococcal organisms (106), yielding a sensitivity and specificity of 100%. The ORTHOProbe system provides a convenient and reliable test for identifying Neisseria gonorrhoeae.

DNA Probes↗

PubMed2003

Automated bead alignment apparatus using a single bead capturing technique for fabrication of a miniaturized bead-based DNA probe array.

We have developed an automated bead alignment apparatus for fabricating a bead-based DNA probe array inside a capillary. The apparatus uses 16 micro vacuum tweezers to extract single beads from among a large amount of beads in bead stock wells. It then manipulates single beads into the probe array capillaries. Single 100-microm-diameter beads were successfully extracted from the water-contained bead-stock well by the vacuum tweezers, which have inner and outer diameters of 50 and 150 microm. An interesting aspect is that unexpected extra beads adsorbed on the outer wall of the vacuum tweezers can be removed using the surface tension force between the water and the atmosphere. In testing the total performance of this apparatus, the DNA probe arrays with 10 sets of probe-conjugated beads and 2 plain beads were produced in the intended order in the capillaries. The time needed to align the 12 beads was 10 min, and the 16 bead arrays were fabricated simultaneously. After hybridization experiments using these fabricated DNA probe arrays, fluorescence from each bead was clearly observed.

Base Sequence↗

PubMedDate not supplied

Use of minisatellite DNA probes for determination of twin zygosity at birth.

Minisatellite DNA probes that detect highly polymorphic regions of the human genome were used to examine DNA from twelve sets of newborn twins. In the seven cases where the twins were known to be monozygotic or dizygotic, from sex observation or placental examination, the DNA result agreed with these findings. In the other five twin pairs and in two sets of triplets DNA analysis allowed rapid determination of zygosity. Minisatellite DNA probes provide a single genetic test that should allow positive determination of zygosity in all cases of multiple pregnancy.

DNA, Satellite↗

PubMed1995

A rapid method to determine bacterial contamination on hatching eggs. 3. Use of commercial DNA probe kits for detection of specific pathogens after six hours of incubation.

The usefulness of commercially available DNA probe kits for the detection of Escherichia coli and Salmonella spp. after only 6 h of incubation, was determined. It was established that the commercially available probe kits used could detect E. coli at initial levels of approximately 4.5 x 10(2) colony-forming units (cfu) per ml after only 6 h of incubation in nutrient broth (NB). Initial bacterial levels as low as 4.5 x 10(1) cfu/ml could be detected when the NB was incubated for 18 h. Salmonella Enteritidis, at initial levels of 2.86 x 10(2) cfu/ml could be detected after 6 h of incubation at 37 degrees C in NB, while initial levels as low as 2.86 x 10(-1) cfu/ml could be detected after 18 h at 37 degrees C in both NB and selected media, as specified by the manufacturers of the probe kits. Commercially available DNA probe kits can therefore be used to detect specific pathogens on the surface of hatching eggs and these probes can be used in conjunction with an egg-washing system, which is used to determine total bacterial contamination, although a longer incubation period greatly improves the sensitivity of these tests.

Animals↗

PubMed1992

Structural chromosome 1 aberrations in transitional cell carcinoma of the bladder: interphase cytogenetics combining a centromeric, telomeric, and library DNA probe.

Fluorescence in situ hybridization (FISH) was used to study numerical and structural chromosome 1 aberrations in interphase nuclei of transitional cell carcinomas (TCCs) of the urinary bladder. One of the characteristic numerical aberrations, as detected previously in low-grade noninvasive TCCs, included trisomy for chromosome 1 (A. H. N. Hopman et al., Cancer Res., 51: 644-651, 1991). We examined in more detail 22 cases with a centromeric (1q12) and a telomeric associated (1p36) DNA probe and with a library DNA probe from sorted human chromosome 1 in single- and double-target FISH procedures. All flow cytometrically determined DNA diploid TCCs (13 cases), which showed three spots for 1q12 (6 cases), had two spots for 1p36. Since the library DNA probe showed three separate domains in the nuclei of these cases, the additional copy for 1q12 could be explained as an extra chromosome 1p-, containing the 1q12 target. In the flow cytometrically determined DNA tetraploid/aneuploid tumors, the results were more complex. In 6 of 9 cases, we observed an overrepresentation of 1q12 as compared to 1p36, also suggesting the presence of extra copies of 1p- chromosomes. The results of the present study demonstrate the utility of the FISH method to assess structural chromosome aberrations in interphase nuclei of solid tumors.

Carcinoma, Transitional Cell↗

PubMed1994

Applications of DNA flow cytometry and fluorescence in situ hybridization using a chromosome-specific DNA probe on paraffin-embedded tissue sections of primary malignant melanomas.

We have applied DNA flow cytometric analysis to paraffin-embedded tissue sections of primary malignant melanomas. Conventionally, flow cytometric analysis of paraffin-embedded tissue sections has been done by the method of Hedley et al. We added ultrasound treatment to the method of Hedley et al. and a lower value of coefficient of variation was shown. Furthermore, a new technique, fluorescence in situ hybridization with a chromosome-specific repetitive DNA probe, was used for the analysis of chromosomal numerical aberrations in the same paraffin-embedded tissue sections. The DNA flow cytometric analysis showed that in 8 cases six primary malignant melanomas were of the aneuploid pattern and two cases of lentigo maligna (melanoma in situ) were of the diploid pattern. By fluorescence in situ hybridization, the two cases with the diploid pattern had spots/nucleus of 1.28 and 1.12, and those with the aneuploid pattern had spots/nucleus from 2.01 to 2.27. Only one nodular melanoma in an aneuploid case showed spots/nucleus of 1.71. These data indicate that fluorescence 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 and may be useful for the study of tumor cell heterogeneity.

Adult↗