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PubMed1985

Comparison of the detection of herpes simplex virus in direct clinical specimens with herpes simplex virus-specific DNA probes and monoclonal antibodies.

A comparison of two commercially available kits for rapid herpes simplex virus (HSV) detection directly in patient specimens was performed. The immunofluorescence assay (IFA) utilized monoclonal antibodies to HSV, and the DNA probe assay utilized three HSV sequences cloned into pBR322. A sample of 243 specimens received in viral transport medium were inoculated into MRC-5 tissue cultures. The remainder of the specimen was centrifuged, and the cellular pellet was examined by IFA and DNA probes. One hundred and sixty-two (66.7%) specimens were considered satisfactory for IFA and DNA probe testing, based on a criterion of observing greater than or equal to 2 intact cells per high-power field. Of the 162 specimens, 35 (21.6%) yielded HSV by culture. By IFA, the sensitivity of detecting HSV culture-positive specimens was 77.1%; specificity was 100%, positive predictive value was 100%, and negative predictive value was 93.3%. DNA probe sensitivity was 71.4%; specificity was 90.6%; positive predictive value was 67.6%; and negative predictive value was 92%. Forty-four (27.2%) of the 162 specimens exhibited nonspecific cytoplasmic staining with the DNA probe. IFA and DNA probe assays can be completed in 2 to 3 h, whereas the average time to culture positivity in this series was 2.2 days. Rapid HSV diagnosis can aid in timely and appropriate patient management.

Antibodies, Monoclonal↗

PubMed1993

Value of a DNA probe assay (Gen-Probe) compared with that of culture for diagnosis of gonococcal infection.

The Gen-Probe PACE 2 system for Neisseria gonorrhoeae (GP), which uses a chemiluminescently labeled DNA probe, was compared with conventional culture as the method of reference. A total of 1,750 specimens were collected from 496 females and 623 males visiting the outpatient clinic of the Sexually Transmitted Diseases Department of the Westeinde Hospital, The Hague, The Netherlands, during the year 1991. The prevalences of gonorrhea culture-positive men and women were 14.9 and 7.7%, respectively. The overall positive rate was 8.7%. Sensitivity, specificity, and positive and negative predictive values of GP were 97.1, 99.1, 90.6, and 99.8%, respectively. A total of 12 of 13 patients with positive GP results and negative cultures may have had a gonococcal infection, a conclusion based on clinical symptoms, positive methylene blue smears, and high relative light unit ratios. The DNA probe test can be useful as a suitable screening and diagnostic test for gonorrheal infection in men and women. An advantage of using this DNA probe technique is that simultaneous testing for Chlamydia trachomatis of the same specimen is possible. We also examined whether (all) rRNA had disappeared after adequate treatment for gonococcal and/or chlamydial infection in 30 patients. None of those positive patients showed a positive result in the DNA probe assay after treatment.

Adolescent↗

PubMed1989

Enzymatic synthesis of DNA probes complementary to a human variable number tandem repeat locus.

Both cloned and synthetic DNA probes complementary to human variable number tandem repeat (VNTR) loci have been used to detect restriction fragment length polymorphism. In this report, we describe an approach for the enzymatic synthesis of a DNA probe complementary to one VNTR locus. The probe is produced by annealing short synthetic oligonucleotides comprising single repeat units and enzymatically ligating them into a polymeric DNA probe. In HinfI digests of human genomic DNA separated by agarose gel electrophoresis, this ligated oligonucleotide probe (LOP) detects multiple polymorphic loci in the range of 3-23 kb producing highly informative DNA fingerprint patterns when different individuals are compared. The hybridization pattern is very stable even under high-stringency wash conditions. The LOP is more easily generated than cloned VNTR probes and is totally synthetic, avoiding problems associated with cloned probes including bacterial growth and maintenance as well as in vitro labeling.

Autoradiography↗

PubMed1987

DNA probes for the detection of mycoplasmas in genital specimens.

The utility of whole-genomic DNA probes for the detection of infections by genital mycoplasmas was investigated in 220 men attending a sexually transmitted diseases clinic. In 144 patients, probe results were compared with quantitative culture results. The prevalence of Mycoplasma hominis was 11% by culture, whereas the prevalence of ureaplasmas was 38%. The M. hominis DNA probe detected 9 of 16 M. hominis culture-positive specimens and 2 of 128 culture-negative specimens. The Ureaplasma urealyticum DNA probe detected 36 of 57 U. urealyticum culture-positive specimens and 18 of 87 culture-negative specimens. Most of the probe-negative culture-positive specimens had colony counts of less than 10(3) organisms/ml of specimen. The DNA probe does not require viable organisms, and the probe-positive, culture-negative specimens suggest that false-negative cultures occurred, perhaps due to specimen handling or insensitivity of culture methods for some strains of mycoplasmas.

Bacteriological Techniques↗

PubMed1996

Comparison of DNA probe (Gen-Probe) with culture for the detection of Neisseria gonorrhoeae in an urban STD programme.

BACKGROUND AND OBJECTIVES: The DNA probe assay is an alternative to culture for the detection of Neisseria gonorrhoeae which does not depend on the viability of the organism. There have been few published studies comparing the two methodologies. The majority of these studies have shown the probe assay to be comparable to culture. The goal of this study was to compare the performance of the DNA probe assay with culture in a high prevalence setting with nearly optimal culture transport conditions. METHODS: Genital specimens for culture and for DNA probe assay were collected at the time of the routine visit for STD clinic patients. Results of the two methods were compared. Discrepant results were further analysed using the results of direct patient Gram stains. RESULTS: Satisfactory matched specimens were available for 999 patients. The overall sensitivity and specificity of the DNA probe as compared with culture was 90 and 96%. Resolved values achieved by factoring in the results of direct patient Gram stains did not significantly improve the performance of the probe. CONCLUSION: Culture remains the test of choice for the detection of gonorrhoea in a high prevalence setting where culture transport conditions are adequate.

Bacteriological Techniques↗

PubMed1990

Comparison of serological and DNA probe analyses for detection of suspected periodontal pathogens in subgingival plaque samples.

Several methods are currently being used to identify specific bacteria in dental plaque, namely direct culture, serological techniques and DNA probes. Culture methods are labour-intensive, dependent on the viability of the cells, and require fastidious growth conditions. Serological and DNA probes allow rapid strain-specific identification of periodontal pathogens with limited effort. The purpose of the present study was to compare results from serological and DNA probes in assessing and quantitating the presence of three suspected periodontal pathogens in subgingival plaque: Actinobacillus actinomycetemcomitans, Bacteroides gingivalis and Bact. intermedius. Subgingival bacterial samples were obtained from 4 periodontal sites of each of 13 patients with untreated moderate to advanced adult periodontitis. Samples were taken using 2 paper points that were simultaneously inserted for 10 s to the bottom of the periodontal pockets. The plaque from one paper point was analysed by immunofluorescence using monoclonal antibodies. The other sample was analysed by species-specific cloned DNA probes. The number of bacteria per millilitre was calculated for both methods, and used for comparisons of results obtained with the two techniques. Results from indirect immunofluorescence and DNA hybridization analyses were negative for A. actinomycetemcomitans across all samples. Fluorescence did not detect bacteria at levels lower than 10(4), while the DNA probes identified organisms at levels of 10(3). Similar numbers of samples positive for Bact. gingivalis were obtained with either method (p = 0.227), and the results were not independent. A significantly greater proportion of Bact. intermedius-positive samples was detected by immunofluorescence (p = 0.0039), and the results of immunofluorescence and DNA hybridization were independent.(ABSTRACT TRUNCATED AT 250 WORDS)

Actinobacillus↗

PubMed1993

[Identification of Mycobacterium tuberculosis complex and Mycobacterium avium-intracellulare complex by using nonisotopic DNA probes].

A total of 31 clinical isolates of mycobacteria consisting of 21 strains of M. avium-intracellulare complex (ten strains each of M. avium and M. intracellulare identified by specific 125I-labeled DNA probes and one strain of M. avium-intracellulare complex identified by conventional biochemical tests) and 10 strains of M. tuberculosis (identified by a specific 125I-labeled DNA probe) were subjected to identification by hybridization protection assay (HPA) with an acridinium-ester (AE) labeled DNA probe. The results thus obtained were in complete agreement with those obtained by 125I-labeled DNA probes (30 strains) or by conventional biochemical tests (one strain). HPA by the AE-labeled DNA probe is safe, simple and rapid, and can be done with ease in any clinical laboratory.

DNA Probes↗

PubMed1992

A comparison of substrates for quantifying the signal from a nonradiolabeled DNA probe.

A method for measuring the amount of a nonradiolabeled DNA probe using four detection substrates is described. In preliminary experiments, digoxygenin-labeled DNA was bound to neutral, nylon membranes and detected with anti-digoxygenin antibodies conjugated to alkaline phosphatase. Four substrates [4-nitrophenyl phosphate, 4-methylumbelliferyl phosphate, AttoPhos, and adamantyl 1, 2-dioxetane phosphate (AMPPD)] were assessed for use in a quantitative hybridization assay. Only AttoPhos and AMPPD were found to have detection limits in the low picogram range and to respond linearly to DNA concentrations ranging from 0 to 1250 pg. In subsequent experiments, a 200-bp DNA probe cloned from the marine bacterium Pseudomonas perfectomarina 23S rRNA gene was hybridized to P. perfectomarina genomic DNA and total RNA. The amount of hybridized probe was determined using AttoPhos. Finally, a digoxygenin-labeled oligonucleotide was probed against genomic DNA. Linearity with respect to DNA concentration was observed using both the 200-bp fragment and the oligonucleotide as probes with a final target detection limit of 166 fg. This study demonstrates the substrate AttoPhos can be used to quantify the amount of nonradiolabeled probe hybridized to target with sufficient sensitivity for very dilute samples, such as environmental samples.

Adamantane↗

PubMed1989

Isolation and mapping of DNA probes within the linkage group I gene cluster of Caenorhabditis elegans.

We have isolated probes for DNA polymorphisms across the linkage group I gene cluster in Caenorhabditis elegans, using Tc1-linkage selection. The probes detect strain polymorphism between the wild-type strains of var. Bristol and var. Bergerac. As a result of mapping the sites hP4, hP5, hP6, hP7, hP9, and sPl, more than 1000 kilobases (kb) of cloned cosmid DNA has been positioned on the genetic map. We found there is more DNA per map unit in the center of the gene cluster than expected on the basis of the genomic average. Furthermore, the amount is not constant across the entire region but reaches a peak in the hP9 unc-13 interval. To find the coding regions, we examined DNA cross-homology between two species, Caenorhabditis elegans and Caenorhabditis briggsae. Approximately one-third of the DNA in the hP5 hP9 interval was examined for coding regions and 21 sequences were identified within 318 kb of DNA.

Animals↗

PubMed1993

Use of a DNA probe for the rapid detection of group B streptococci in obstetric patients.

OBJECTIVE: To determine the accuracy of a DNA probe as a rapid diagnostic test for detecting colonization of the female genital tract by group B streptococci during pregnancy. METHODS: Two rayon-tipped applicators were used to collect secretions from the posterior vaginal wall of 440 pregnant women. One of the applicators was inoculated into selective Todd-Hewitt broth and used as the reference standard for identification of group B streptococci. The other applicator was used for analysis with the DNA probe, preceded by either 2.5 hours of incubation for the initial 75 patients, or 3.5 hours' incubation for the remaining women. Following hybridization with an acridinium-labeled probe, chemiluminescence was measured with a luminometer. RESULTS: The prevalence of positive cultures was 20%. For the initial 75 patients whose cultures were amplified by incubation for 2.5 hours, the DNA probe had a sensitivity of 44%, specificity 94%, positive predictive value 79%, and negative predictive value 77%. For the cultures that were incubated for 3.5 hours, respective values were 71, 90, 61, and 94%. All vaginal specimens that had an average initial cell count of 1.5 x 10(3) cells/mL were accurately detected by the probe after 3.5 hours' growth amplification. False-positive results occurred primarily when the specimens were grossly contaminated with blood (26 of 39). The mean time required to perform the assay, including 3.5 hours of growth amplification, was 4.3 hours. CONCLUSIONS: The DNA probe demonstrated good overall sensitivity and gave no false-negative results when group B streptococci were present in concentrations of 1 x 10(4) cells/mL or greater. Sensitivity improved significantly with 3.5 hours' growth amplification as compared with 2.5 hours (P < .05), reflecting better identification of lightly colonized patients.

Carrier State↗

PubMed1992

[Cytogenetic analysis of lung tumors by in situ hybridization with chromosome-specific DNA probes].

Fluorescent in situ hybridization (FISH) with biotinated chromosome-specific repetitive DNA probes was used for the cytogenetic study of lung tumors and three cell lines of human lung cancer. The authors utilized a set of satellite DNA probes, specific for chromosomes 7, 17, X, Y in order to detect numerical chromosome aberrations in tumor cell nuclei. Normal diploid human lymphocyte nuclei, which served as the control, have have two signal spots in 95% of nuclei in response to 7, 17 chromosome probes. However, lung cancer cells have numerical heterogeneity, and copy numbers as determined by FISH were not definite with each probe. Discrepancies between cytogenetic and flow cytometric studies in the detection of aneuploidy in some tumors were shown. The number of FISH spots showed a correlation only with the Ki-67 labeling index expressed in proliferating cells. Loss of the Y chromosome in a high percentage of cells was seen by FISH in some tumors from male patients. These data indicate that FISH with chromosome-specific repetitive DNA probes can serve as a cytogenetic tool for the analysis of interphase nuclei of lung tumors with respect to the detection of numerical chromosome abnormalities.

Adult↗

PubMed1987

A cross-reactivity study of whole genomic DNA probes for Haemophilus actinomycetemcomitans, Bacteroides intermedius, and Bacteroides gingivalis.

In this study, we evaluated the sensitivity and specificity of whole genomic DNA probes for the periodontal pathogens Haemophilus actinomycetemcomitans, Bacteroides intermedius, and Bacteroides gingivalis. By means of these probes, DNA hybridizations were performed against other organisms found in the oral cavity and organisms previously determined to be genetically similar. All three probes were sensitive to 10(3) cells for their respective organism. The H. actinomycetemcomitans probe cross-reacted with several haemophilus strains, Wolinella, and Campylobacter, indicating that H. actinomycetemcomitans-specific sequences would have to be identified and cloned for accurate detection of this organism in heterogeneous patient samples. Only very low levels of cross-reactivity were observed between the B. intermedius probe and representative black-pigmented Bacteroides. This low level of cross-reactivity did not interfere with the accurate identification of B. intermedius in sample evaluations. The B. gingivalis probe showed no cross-reactivity. Whole genomic probes will be used for the detection of B. intermedius and B. gingivalis in patient samples.

Bacteroides↗

PubMed1991

Diagnostic applications of DNA probes.

This review has described several of the most common molecular biologic techniques that are, or will be, employed in the diagnostic laboratory. The potential advantages of these DNA probe assays in the diagnosis of infectious diseases include: rapid detection and identification of infectious agents; the ability to screen selected specimens using batteries of probes; and the detection of nonviable or difficult-to-culture organisms. The potential disadvantages of DNA probe assays include: the use of isotopic detection methods for optimum sensitivity; limited diagnostic sensitivity of current assays; slow turna-round time for some assay formats; expense of current reagents; limited availability of many probes; lack of technical expertise in most diagnostic laboratories; and the requirement for antimicrobial susceptibility testing (requires culture). Given the above advantages and disadvantages, there are several key issues that must be considered before adopting DNA probe technology in the diagnostic laboratory; the cost of performing routine culture and identification versus the cost of screening with probes--both the number and type of specimens and the time savings that may be realized by eliminating routine cultures; the prevalence of the infectious agent--even the best DNA probe assay may not be useful or practical in a low-prevalence situation; the need for additional equipment and space; and the interpretation of false-positive and false-negative results--additional research is needed in this area. However, laboratories must consider these issues when using a test other than the current gold standard (i.e., culture). DNA probe technology is with us and expanding rapidly. The intelligent application of this new technology will require communication between laboratorians and clinicians and careful consideration of the many advantages and disadvantages discussed above.

Blotting, Southern↗

PubMed1988

Detection of viral DNA by in situ hybridization using bromodeoxyuridine-labeled DNA probes.

Detection of viral DNA in infected tissues is demonstrated by in situ nucleic acid hybridization employing bromodeoxyuridine (BrdUrd)-labeled probes. BrdUrd is used alternatively to biotinylated nucleotides in the preparation of DNA probes and, after hybridization, developed with a monoclonal anti-BrdUrd antibody and the alkaline phosphatase-anti-alkaline phosphatase technique. The sensitivity using both BrdUrd- and biotin-labeled probes is similar. Furthermore, the simultaneous detection of viral coinfections will be possible by means of this technique in conjunction with biotin-labeled DNA probes.

Bromodeoxyuridine↗

PubMed1989

Use of a biotinylated DNA probe to detect bacteria transduced by bacteriophage P1 in soil.

Presumptive bacteriophage P1 transductants of Escherichia coli, isolated from soil inoculated with lysates of transducing phage P1 and E. coli, were confirmed to be lysogenic for phage P1 by hybridization with a biotinylated DNA probe prepared from the 1.2-kilobase-pair HindIII 3 fragment of bacteriophage P1. No P1 lysogens of indigenous soil bacteria were detected with the DNA probe. The sensitivity and specificity of the DNA probe were assessed with purified and dot blot DNA, respectively. In addition, two techniques for the lysis and deproteinization of bacteria and bacteriophages on nitrocellulose filters were compared. These studies indicated that biotinylated DNA probes may be an effective alternative to conventional radiolabeled DNA probes for detecting specific gene sequences in bacteria indigenous to or introduced into soil.

Bacteria↗

PubMed1993

[The use of DNA probes for the diagnosis of pulmonary tuberculosis].

Mycobacterium-specific nonradioactive DNA probes have been tested able to detect mycobacteria in clinical samples (sputum, bronchoalveolar lavage) for 48 hours. The probes sensitivity makes up 1000-10,000 cells. Molecular hybridization confirms the results of DNA-probe diagnostic conclusion of tuberculosis more frequently than the results of standard microbiological tests.

Adult↗

PubMed1998

The development of random DNA probes specific for Aeromonas salmonicida.

RAPD-PCR has been used to produce DNA probes for Aeromonas salmonicida. DNA hybridization studies showed that RAPD-PCR fragments of the same size did not necessarily hybridize to each other and therefore these sequences were not always homologous. However, a single RAPD-PCR fragment (designated 15e) was identified as being common to Aer. salmonicida. Subsequently, 15e was found to comprise five DNA fragments of similar size which differed in their nucleotide sequences. All five fragments were evaluated as DNA probes for the specific detection of Aer. salmonicida DNA: two hybridized specifically to DNA of all Aer. salmonicida isolates tested, including the four current subspecies and atypical isolates; one hybridized to subspecies salmonicida, achromogenes and masoucida, but not subspecies smithia; one hybridized to subspecies salmonicida and achromogenes, but not subspecies masoucida or smithia; and one hybridized to subspecies salmonicida, achromogenes and smithia, but not subspecies masoucida. It is believed that these fragments could be useful as non-radioactive probes for the safe and rapid diagnosis of these fish pathogens.

Aeromonas↗

PubMed1992

Identification of Selenomonas species by whole-genomic DNA probes, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, biochemical tests and cellular fatty acid analysis.

Nonisotopic, whole-genomic DNA probes, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), biochemical tests in microtiter trays and cellular fatty acid (CFA) analysis were compared for the identification of 5 oral Selenomonas species. DNA probes were prepared by biotin-labeling DNA extracted from the type strains of Selenomonas noxia, Selenomonas flueggei, Selenomonas artemidis, Selenomonas infelix and Selenomonas sputigena. The probes were hybridized with DNA from 21 reference strains, 18 fresh isolates of Selenomonas species, and 21 strains of other oral gram-negative species. Target DNAs were obtained by in situ extraction of colonies blotted onto filter paper. Streptavidin-linked alkaline phosphatase was used to detect homologous reactions of probe and target DNA. Each Selenomonas species DNA probe reacted with reference strains of only that species. All Selenomonas strains that reacted with the DNA probe for a particular species gave similar biochemical test results, SDS-PAGE protein profiles, and CFA profiles to those of the type strain of the corresponding species. All the methods tested were useful for identifying the species, and all yielded similar identifications of the fresh isolates. The DNA probes, however, had the potential for identifying Selenomonas species directly from primary isolation plates or plaque samples.

Bacteroidaceae↗