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At least 19 recordsLinked to original sources
PubMed1991

Comparison of Gen-Probe DNA probe test and culture for the detection of Neisseria gonorrhoeae in endocervical specimens.

A 2-h nonisotopic DNA probe assay for the direct detection of Neisseria gonorrhoeae in urogenital specimens has recently been modified (PACE 2; Gen-Probe, San Diego, Calif.). The new assay format was developed to increase the sensitivity of the assay and simplify procedural steps. In this study, the new DNA probe test was compared with a culture reference method for the detection of N. gonorrhoeae in endocervical specimens. The results of the DNA probe test were expressed as a ratio of relative light units (RLU) of the specimen/RLU of the cutoff recommended by the manufacturer. All patient samples with sample RLU/cutoff RLU ratios less than 0.7 were interpreted as negative, and ratios greater than 2.0 were interpreted as positive for gonorrhea. Samples with sample RLU/cutoff RLU ratios between 0.7 and 2.0 were repeated until two or more consistent negative or positive ratios were obtained. A total of 469 specimens were tested with an overall disease prevalence of 6.1%. Of the 469 patients tested, 5 specimens (1.0%) fell in this borderline region and were retested. If the manufacturer's recommended cutoff value had been used, the original DNA probe results would have resulted in two false-positives. Our data were analyzed for both symptomatic (prevalence, 11.7%) and asymptomatic (prevalence, 2%) women. The study indicated that with our modification of the manufacturer's endpoint interpretation, the DNA probe test was essentially equivalent to the culture method in terms of sensitivity, specificity, and positive and negative predictive values in both symptomatic and asymptomatic patient populations. The new DNA probe test can serve as a suitable screening and diagnostic test for the diagnosis of gonorrheal genital infections in women. Additionally, it offers the advantages of rapid turnaround time and ease of use and allows simultaneous testing for Chlamydia trachomatis on the same specimen.

Bacteriological Techniques↗
PubMed1993

Comparison of "Gen-Probe" DNA probe and PCR for detection of Listeria monocytogenes in naturally contaminated soft cheese and semi-soft cheese.

A comparison was made of three approaches for the detection of Listeria monocytogenes in naturally contaminated soft cheese and semi-soft cheese after enrichment. Enrichment broths were tested by plating them onto different selective agars, by "Gen Probe" DNA hybridization and by the polymerase chain reaction (PCR). Based on two-step enrichment, all three approaches showed high specificities (90% or more) in detecting L. monocytogenes. In contrast, the sensitivity of the Gen-Probe test was low (33% or less), whereas high sensitivities were obtained with selective plating and PCR (83% or more). Based on one-step enrichment, specificities again were high for selective plating and PCR assay (100%), whereas for the Gen-Probe assay the specificity was lower (88% or more). The best sensitivities were observed with selective plating (67%) and PCR (75%). In terms of sensitivity, specificity and analysis time, PCR applied to a two-step enrichment was the most powerful assay for detecting L. monocytogenes in soft and semi-soft cheese.

Cheese↗
PubMed1986

Evaluation of the Gen-Probe DNA probe for the detection of legionellae in culture.

A commercial DNA probe kit designed to detect rRNA from legionellae was evaluated for its ability to correctly discriminate between legionellae and non-legionellae taken from culture plates. The probe kit, made by the Gen-Probe Corp. (San Diego, Calif.), was radiolabeled with 125I, and probe bacterial RNA hybridization, detected in a simple one-tube system hybridization assay, was quantitated with a gamma counter. A total of 156 Legionella sp. strains were tested, of which 125 were Legionella pneumophila and the remainder were strains from 21 other Legionella spp. A total of 106 gram-negative non-legionellae, isolated from human respiratory tract (81%) and other body site (19%) specimens, were also tested; 14 genera and 28 species were represented. The probe easily distinguished all of the legionellae from the non-legionellae. The average legionellae/non-legionellae hybridization ratio was 42:1, and the lowest ratio was 2:1; a minor modification in the procedure increased the lowest ratio to 5:1. In addition to correctly identifying all Legionella species, the probe was able to separate some of the various species of Legionella. L. pneumophila strains hybridized more completely to the probe than did the other Legionella spp.; L. wadsworthii and L. oakridgensis hybridized only about 25% of the probe relative to L. pneumophila. Some strains of phenotypically identified L. pneumophila had much lower hybridization to the probe than other members of the species and may represent a new Legionella species. The simplicity of the technique and specificity of the probe make it a good candidate for confirming the identity of legionellae in culture.

DNA↗
PubMed1994

[Detection of Campylobacter species by using polymerase chain reaction and nonradioactive DNA probes. III. DNA probe for identification of C. laridis].

The development of a rapid and specific DNA probe assay for identification of Campylobacter species, including C. jejuni, C. coli, C. laridis, C. fetus, and C. hyointestinalis is important in determining the precise diagnosis of Campylobacter infections. Sequence data of our previous studies for a 240-base DNA fragment was used to select primers and probes conjugated to alkaline phosphatase, complementary to a portion of DNA between primers. However, a 21-base probe (CL (1)) tested here for detection of C. laridis was cross-reactive with PCR-amplified fragments of C. jejuni, C. coli and C. hyointestinalis, although it was not reactive with C. fetus and C. fetus subsp. fetus. To solve this problem, further modifications of the probe were therefore made to improve the specificity for those particular species. A second 21-base probe with a single base-substitution (CL (2)) and a third 20-base probe (CL(3)) were ineffective for identification of C. laridis, too. A fourth 20-base probe with a single base substitution (CL(4)) was a significant improvement over the results obtained by other three probes specifically to detect C. laridis, Thus, the alkaline phosphatase-labeled probe method developed so far is an interesting alternative without access to radioisotopes for clinical laboratories for identification of Campylobacter species, including C. jejuni/coli/hyointestinalis, C. laridis, and C. fetus/fetus subsp. fetus.

Base Sequence↗
PubMed1991

The characterization of Gardnerella vaginalis DNA using non-radioactive DNA probes.

DNA restriction profiles of various Gardnerella vaginalis isolates, generated by BamHI, EcoRI, PstI and other restriction enzymes, varied considerably. Only a few DNA fragments were identified as common in ethidium bromide fluorescence profile and Southern-blot hybridization patterns (employing a digoxigenin-labelled G. vaginalis DNA probe and an enzyme-linked immunoassay detection method). While the efficiencies of Southern-blot hybridization appeared inconsistent, in dot-blot assays, DNA from each isolate hybridized readily, enabling the detection of at least 10 ng DNA. A 5.7-kb DNA fragment from G. vaginalis ATCC 14018 genomic library, cloned in the BamHI site of pBR322, could replace the total genomic DNA probe. This specific DNA fragment was present in different sizes in 12 analysed G. vaginalis strains, describing a restriction fragment length polymorphism. In control studies, none of the DNA from bacteria other than G. vaginalis (including some genitourinary tract residents) hybridized with the G. vaginalis total or specific DNA probes. Non-radioactive G. vaginalis DNA probes can thus form the basis of a useful detection method for further studies of this organism.

Blotting, Southern↗
PubMed1989

Direct identification of Mycobacterium tuberculosis, Mycobacterium avium, and Mycobacterium intracellulare from amplified primary cultures in BACTEC media using DNA probes.

DNA probes (Gen-Probe, San Diego, Calif.) directed at the Mycobacterium tuberculosis complex and Mycobacterium avium-M. intracellulare complex were used to identify acid-fast bacilli directly from specimens grown in BACTEC 12B bottles (Becton Dickinson and Co., Towson, Md.). Clinical specimens were inoculated directly or after decontamination into a BACTEC 12B bottle, Middlebrook 7H11 agar, and Lowenstein-Jensen medium. Conventional media were incubated at 37 degrees C in 5% CO2 and examined weekly for 6 weeks. Identification of isolates grown on conventional media by standard biochemicals, morphology, and growth characteristics served as the reference method for identification. BACTEC bottles were incubated at 37 degrees C, and a growth index was taken twice a week. When a growth index of greater than or equal to 100 was reached, 1 ml of BACTEC 12B medium was put into each of three microfuge tubes which were centrifuged for 15 min at 15,000 x g. Pellets were used in hybridization reactions with an M. tuberculosis complex probe, an M. avium probe, and an M. intracellulare probe. The results of the hybridizations of the three probes with the same sample were compared, and the highest percent hybridization was divided by the average of the lower hybridization values. If this value, the derived patient ratio (DPR), was greater than or equal to 3, then the specimen was considered positive for the organism giving the highest percent hybridization. Of the 1,988 specimens cultured, the results of conventional tests for the 190 conventional culture-positive specimens were 64 M. tuberculosis, 61 M. avium, 14 M. intracellulare, 30 other Mycobacterium spp., and 25 non-acid-fast bacilli. There were four cultures that each contained two different Mycobacterium spp. Directly probing the BACTEC 12B sediment, at DPR of >/= 3 the M. tuberculosis probe identified 83% (53 of 64) of M. tuberculosis isolates, the M. avium probe identified 92% (56 of 61) M. avium isolates, and the M. intracellulare probe identified 86% (12 of 14) of M. intracellulare isolates. There were no false-positive results at this DPR level. The false-negative results from probing the sediment from the BACTEC 12B bottle could not solely be attributed to the number of organisms present, the growth index, or antimicrobial therapy.

Colony Count, Microbial↗
PubMed1992

DNA fingerprinting of Chlamydia trachomatis by use of ribosomal RNA, oligonucleotide and randomly cloned DNA probes.

DNA fingerprinting of 15 reference strains and 24 clinical isolates of Chlamydia trachomatis, 2 strains of C. psittaci and one strain of C. pneumoniae was studied by use of universal 16 + 23S RNA from Escherichia coli, 16S rDNA-directed oligonucleotide and randomly cloned chlamydial DNA probes. The rRNA-gene restriction patterns (ribotypes) enabled the differentiation of chlamydial species. Following DNA cleavage by restriction endonuclease PvuII, lymphogranuloma venereum and trachoma biovars of C. trachomatis could be differentiated. An oligonucleotide, designed to hybridize the C. trachomatis 16S rDNA, also allowed for both species-specific identification and biovar typing of C. trachomatis human strains. Molecular typing system using 3 lambda clones containing C. trachomatis serotype E random DNA inserts, combined to ribotyping, revealed 12 groups of variable banding patterns within C. trachomatis, and could provide an alternative epidemiological tool.

Autoradiography↗
PubMed1991

Comparison of colony and stool blots for detection of enteropathogens by DNA probes.

DNA probes that identify genes coding for heat-labile type I (LT-I) and heat-stable type 1 (ST-I) enterotoxins, enteropathogenic Escherichia coli adherence factor (EAF), and Shigella-like invasiveness (INV) are used to evaluate the sensitivity and specificity of stool blots in comparison with the sensitivity and specificity of colony blots in detecting enteropathogens. The sensitivities of the probes in stool blots are 91.7% for the LT-I probe, 76.9% for the ST-I probes, 78.9% for the EAF probe, and 45.5% for the INV probe. The specificity of all probes is higher than 95%. In general, the stool blot method identifies as many if not more LT-I-, ST-I-, and EAF-producing E. coli infections than the colony blots.

Adhesins, Escherichia coli↗
PubMed1993

Genetic diversity of Helicobacter pylori indexed with respect to clinical symptomatology, using a 16S rRNA and a species-specific DNA probe.

DNA probes are described which identify group and fingerprint strains of the human gastric pathogen Helicobacter pylori, on the basis of well-defined band homologies. A 544 bp internal fragment of the 16S ribosomal RNA gene was generated by polymerase chain reaction (PCR) with primers derived from the Escherichia coli rRNA gene sequence. In genomic Southern blots this probe detected restriction site variation around these loci, generating simple but strain-specific molecular fingerprints. A small conserved chromosomal fragment of 1.2 kbp, Hps, species-specific for H. pylori, was obtained by cloning random HindIII fragments into pUC19. It was useful for dot-blot identification, and also separated isolates into one major and two minor groups. When results for these two probes were combined, a baseline characterization of genotype was obtained. A band-matching database of molecular fingerprints for the type strain and 63 clinical isolates of H. pylori from asymptomatic, ulcer and gastritis contexts is presented. No significant association between the genotypes at this level of definition and the associated clinical symptomatology of the isolates was detected.

DNA Primers↗
PubMed1987

Classification of Campylobacter strains using DNA probes.

DNA homology determinations were used to classify atypical strains of Campylobacter spp. and Campylobacter-like organisms. Using the slot-blot hybridization technique, atypical strains were classified as C. coli or C. jejuni. Some of the Campylobacter-like organisms isolated from animals were catalase-positive, but they did not hybridize with the DNA probes prepared from the total genomic DNA of C. coli, C. jejuni, C. laridis, C. fetus subsp. fetus and C. hyointestinalis. The taxonomic classification of these strains remains uncertain. The slot-blot hybridization technique was simplified by applying whole cells from a 24 h broth culture, instead of pure DNA to nitrocellulose membranes. The results of DNA homology from both methods were comparable, and the classification of these organisms using these methods was the same. Therefore, the dot-blot hybridization method is recommended as a more economical and time-saving method for use in the taxonomic classification of atypical Campylobacter spp.

Base Composition↗
PubMed1986

Enumeration of polysaccharide-degrading Bacteroides species in human feces by using species-specific DNA probes.

DNA probes that are specific for each of five predominant species of human colonic Bacteroides (B. thetaiotaomicron, B. uniformis, B. distasonis, "Bacteroides group 3452-A", and B. ovatus) were used to detect and enumerate these species in fecal samples from two adult volunteers. These five species are capable of fermenting many of the complex polysaccharides that are thought to be sources of carbon and energy for bacteria in the colon. Estimates for the concentrations of some of these species in feces have not been previously available because of the difficulties in differentiating colonic Bacteroides spp. by conventional biochemical tests. Our results indicate that all the species except B. ovatus were present in high numbers (greater than 10(9)/g [dry weight]) in the feces of both volunteers. However, the concentrations of the more versatile polysaccharide-degrading species within this group of organisms (7.6 X 10(9) to 12.0 X 10(9)/g [dry weight] for B. thetaiotaomicron; 2.9 X 10(9) to 6.3 X 10(9)/g [dry weight] for "Bacteroides group 3452-A") did not differ significantly from the concentrations of less versatile polysaccharide-degrading species (1.2 X 10(10) to 2.0 X 10(10)/g [dry weight] for B. uniformis; 5.8 X 10(9) to 8.4 X 10(9)/g [dry weight] for B. distasonis). B. ovatus was not detectable by our method. Since our lower limit of detection is approximately 1 X 10(9) to 2 X 10(9)/g (dry weight) of feces, this is consistent with earlier estimates that indicated that the concentration of B. ovatus in feces is near or below this value.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacteroides↗
PubMed1989

Development of a rapid method for determining the susceptibility of Mycobacterium tuberculosis to isoniazid using the Gen-Probe DNA Hybridization System.

A rapid test was developed for determining the susceptibility of Mycobacterium tuberculosis to isoniazid by using nucleic acid hybridization. The method was based on quantification of total mycobacterial rRNA hybridized to a 125I-labeled DNA probe in the absence and presence of various concentrations of isoniazid. The radioactive hybridized complex was isolated by adsorption to hydroxyapatite crystals and measured in a gamma counter. The susceptibilities of four reference strains and 20 clinical isolates were compared by the Gen-Probe DNA Hybridization System and the critical concentration method. Overall agreement between the two methods was excellent. Results were obtained with the DNA probe after 3 to 5 days of incubation instead of the 14 to 21 days required for the critical concentration method. These findings indicate that susceptibility testing of M. tuberculosis by nucleic acid hybridization has merit for the clinical laboratory. Additional studies are needed to determine the efficacy of the DNA probe method for determining the susceptibility of M. tuberculosis to other antimycobacterial agents and its correlation with clinically significant levels of resistance.

Culture Media↗
PubMed1995

Prior enrichment of human immunodeficiency virus DNA with probe DNA particles for efficient PCR diagnosis.

We report here a novel method by which one can recover human immunodeficiency virus (HIV) DNA in a small volume (approximately 50 microliters) of solution from 8 ml of crude cell lysate which contains as few as several HIV DNA copies. The method uses the specific binding of HIV DNA to HIV probe DNA particles. The HIV DNA thus concentrated on the particles can be subjected to the PCR assay. The method also enables the examination of 100 individual blood specimens in a combined form.

Base Sequence↗
PubMed1993

Radiation-induced chromosome aberrations analysed by two-colour fluorescence in situ hybridization with composite whole chromosome-specific DNA probes and a pancentromeric DNA probe.

Fluorescence in situ hybridization with composite whole chromosome-specific DNA probes for human chromosomes 1, 4 and 12 and a degenerate alpha-satellite pancentromeric DNA probe labelled with digoxigenin was used to measure symmetrical translocations and dicentrics induced in vitro by 137Cs gamma-rays (0-6.0 Gy) in peripheral lymphocytes. Despite subtracting our mean background translocation frequency of 0.0016 per cell (11,411 cells scored from 11 individuals) from induced frequencies, about 1.3-1.8-fold more translocations were found than dicentrics at a given dose. Translocation frequencies determined only in stable cells agree well with total translocation frequencies determined also in cells containing additional unstable chromosomal changes. The linear quadratic calibration curve generated for total stable translocations is based on approx. 17,000 cells. The suitability of this curve for biological dosimetry of human radiation exposure can now be evaluated in comparison with dose estimates based on a conventional dicentric dose-response curve.

Cesium Radioisotopes↗
PubMed1994

[Rapid identification of mycobacteria by combined method of polymerase chain reaction and the gen-probe DNA hybridization system].

We developed the rapid detection and identification method of mycobacteria, involving amplification of mycobacterial 16S rRNA gene by nested PCR and identification of M. tuberculosis complex or M. avium-intracellulare complex (MAC) by hybridization protection assay (HPA) using the acridinium-ester (AE) labeled DNA probe. The specificity of the nested PCR combined with DNA probe test was excellent in terms of detection of mycobacterial organisms and identification of M. tuberculosis or MAC. The detection limits of the present method were 10 fg DNA for M. tuberculosis, and 100 fg DNA for MAC, respectively. We further investigated on the optimum temperature for hybridization in HPA with AE labeled DNA probe because there was the difference in the mode of DNA-RNA hybridization from that of DNA-DNA hybridization. In our method, the optimum temperature of hybridization was estimated as 55 +/- 1 degrees C. In preliminary experiments on two clinical cases, we practically detected and identified M. tuberculosis and MAC in clinical specimens, such as sputa, by using this newly devised method. We concluded that this method is useful for rapid detection and identification of M. tuberculosis and MAC in clinical specimens.

Adult↗
PubMed1985

Detection of Escherichia coli adhesins with DNA probes.

DNA hybridization assays for genes encoding the Escherichia coli adhesins K88 and K99 were developed. These assays were used to screen a variety of animal E. coli strains, and the results were compared with results obtained by serological methods. All methods compared well for determining the presence of adhesin K99. However, the DNA hybridization detected more K88-positive strains than did either enzyme immunoassay or slide agglutination. Agreement was improved when strains were grown on blood or nutrient agar instead of Minca medium.

Adhesins, Escherichia coli↗
PubMed1995

[Prior enrichment of HIV-DNA with probe-DNA particles for an efficient PCR diagnosis].

PCR mediated detection of HIV-DNA has been widely used. However, compared with traditional immunological diagnoses, the extraction of DNA is a laborious and time consuming step. We have developed a procedure for the efficient isolation and concentration of HIV-DNA from cell lysates. We report here a novel method by one can recover HIV-DNA in a small volume (approximately 50 microliters) of solution from a large volume of crude cell lysate which contains as few as several copies. The method uses the specific hybridization of HIV-DNA to HIV probe-DNA particles. This prior enrichment augmented the sensitivity in the detection of HIV-DNA by PCR, and allows us to make a diagnosis even if the specimen contained an extremely low copy number of HIV-DNA molecules in a large volume, which would have otherwise resulted in false-negative data with the conventional extraction method. The method also enables the examination of 100 individual blood specimens in a combined form. Thus, the application of the present enrichment procedure with HIV probe-DNA particles should reduce the labor and cost of HIV diagnosis, since the HIV positive samples represent a very minor group of people among specimens subjected to clinical laboratory tests, and particularly, among blood samples voluntarily donated to be used for transfusions.

Base Sequence↗
PubMedDate not supplied

Comparison of three techniques for concentrating positive BACTEC 13A bottles for mycobacterial DNA probe analysis.

Three methods of concentrating 1 ml aliquots from BACTEC 13A bottles containing patient blood samples were evaluated for testing with the Gen-Probe Rapid Diagnostic System for Mycobacteria avium complex: 1. using no reagents, 2. using both lysing and wash reagents; and 3. using lysing reagent only. Aliquots from 13As containing human blood and seeded with eight mycobacterial species were also concentrated directly and using both reagents. Results for samples containing M. avium were as follows: 1. using the direct concentration technique 34 of 47 samples (72%) gave unequivocally positive results; 2. 43 of 47 samples (92%) concentrated using both reagents gave positive results; 3. the technique using lysing reagent only was not found useful. There were no false positives with any of the seeded specimens. We were also able to define the minimum Growth Index necessary to ensure un-equivocally positive results for each concentration technique. For those samples containing M. avium these values were 42 for the technique using both reagents and 86 for the direct technique. Direct or reagent concentration of 13A aliquots for testing with Gen-Probe DNA probes provides a rapid, sensitive, and specific means for the identification of M. avium complex bacteremia.

Bacteriological Techniques↗