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PubMed1987

Immunological capture of nucleic acid hybrids and application to nonradioactive DNA probe assay.

Antibodies specific for DNA:RNA hybrids were coated onto polystyrene test tubes and applied to hybridization assays involving DNA and RNA. Synthetic DNA probes complementary to 16S rRNA of Campylobacter were labeled with biotin and hybridized to ribosomal RNA directly in lysates of bacterial cells. After hybridization, DNA:RNA hybrids were captured with immobilized anti-DNA:RNA antibody, and the biotinylated probe was detected with streptavidin-horseradish peroxidase (EC 1.11.1.7) conjugate. The assay was optimized to detect as few as 70,000 Campylobacter cells in a sample. We compared the utility of this hybridization assay with that of conventional microbiology methods by examination of 1448 stool samples from hospital clinical laboratories. The DNA hybridization assay had a sensitivity of 98.7% (75/76) and a specificity of 98.2% (1347/1372) and overall agreed with 98.2% of the conventional results for a test population that had a 5.2% incidence (76/1448) of Campylobacter infection. The assay is simple to perform and yields results within 2.5 h.

Antibodies↗

PubMed1992

Modified spot hybridization test using biotinylated DNA probe.

A modification that simplifies the spot hybridization technique is described for using biotinylated DNA probes. Plasmid EWD299 having LT gene insert, labelled with biotin either by nick translation or using photobiotin was used as DNA probe for the specific detection of enterotoxigenic Escherichia coli. A simple protocol has been described for easy lysis of test samples by boiling in distilled water followed by detergent treatment and was found to be as efficient as the lysis using lysozyme and protease. Three different solid supports namely DEAE-cellulose paper, nitrocellulose paper and nylon membrane were also compared for their suitability in this spot hybridization test. Nitrocellulose paper was found to give better colour signal with the photobiotinylated DNA probe.

Biotin↗

PubMed1993

Design of Onchocerca DNA probes based upon analysis of a repeated sequence family.

Repeated DNA sequences have been instrumental in the development of DNA probes for many different parasites. Isolation of such DNA probes has generally been accomplished by differential screening of genomic libraries with total genomic DNA preparations. In the current work, a rational design strategy is presented for the development of oligonucleotide probes based upon repeated sequence families. A repeated sequence family present in the genome of Onchocerca parasites, designated O-150, has been amplified from various samples of genomic DNA using PCR. DNA sequence analysis of the resulting PCR products demonstrated that the sequences may be arranged into clusters within which the individual sequences are identical or nearly identical. Differences among the cluster consensus sequences have been exploited to explain the specificities of previously isolated O-150 based probes and to develop two new oligonucleotide probes. One of these probes hybridizes specifically to Onchocerca volvulus O-150 PCR products, while the second hybridizes specifically to O-150 PCR products from the closely related bovine parasite O. ochengi. These oligonucleotide probes have been used to characterize Onchocerca infective larvae isolated from wild caught infected flies in West Africa. Because repeated sequence families are a common feature of most genomes, including those of parasites, this method should be applicable to the rational design of oligonucleotide probes for other parasitic infections.

Animals↗

PubMed1985

First trimester prenatal diagnosis and detection of carriers of haemophilia A using the linked DNA probe DX13.

Although the use of a gene specific deoxyribonucleic acid (DNA) probe is the method of choice for detecting carriers of genes for rare genetic disorders, there will always be families in which such probes cannot be used because key subjects are not informative for restriction fragment length polymorphisms in or around the gene. In these cases closely linked DNA markers have to be used. An X chromosome specific DNA probe, DX13, which is closely linked to the haemophilia A locus on the X chromosome, was used for early prenatal diagnosis in two cases and to detect carriers in a series of nine possible heterozygote women. The first reported crossover between DX13 and the factor VIII:C locus was observed in this study. There are complexities inherent in using any linked DNA probe for assignment of genes, but such techniques are clinically important.

Female↗

PubMed1999

Comparison of DNA probe technology and automated continuous-monitoring blood culture systems in the detection of neonatal bacteremia.

OBJECTIVE: The present study assessed the ability of a rapid chemiluminescent DNA probe assay to detect bacterial growth in blood culture specimens before their detection by continuous-monitoring blood culture (CMBC) systems. STUDY DESIGN: Three newborn intensive care units, which are members of the National Institute of Child Health and Human Development Neonatal Research Network, participated in this study. Each center employs an automated CMBC system against which the DNA probe was compared. A total of 1700 blood cultures were analyzed. A 3-ml aliquot of culture medium was removed from each culture during early incubation, processed, and analyzed by the DNA probe. RESULTS: Of 1700 blood cultures, 130 (7.6%) were detected positive by a CMBC system. Coagulase-negative staphylococci (CNS) were present in 90 (69%) of the positive cultures, and 26 (29%) were detected by the DNA probe. Other organisms accounted for the remaining 40 positive cultures, and 31 (78%) of these were detected by the probe assay. Seventy-six percent of all positive cultures were detected by a CMBC system within 24 hours. Ninety-eight percent of all positive cultures were detected by a CMBC system within 48 hours. Of the two organisms that grew in a CMBC system beyond 48 hours, both were CNS and one of these was considered a contaminant. Therefore, 99% of clinically significant organisms were detected by a CMBC system within 48 hours. CONCLUSION: This DNA probe is not sufficiently sensitive to be clinically useful; however, automated CMBC systems are sufficiently sensitive to aid in clinical decision-making regarding the continuance or discontinuance of antibiotic therapy following 48 hours of culture incubation.

Bacteremia↗

PubMed1994

Use of non-radioactively labeled DNA probes for the detection of a baculovirus from Penaeus monodon by in situ hybridization on fixed tissue.

Clones isolated from a genomic library of the baculovirus PmSNPV (= MBV) were used to prepare DNA probes for detection of PmSNPV in shrimp tissue. Davidson's AFA fixed shrimp tissues were probed using DNA that had been labeled with the hapten, digoxigenin, by an in situ hybridization assay. The DNA probes readily distinguished PmSNPV-infected from uninfected shrimp tissue. The reactions were localized to the hepatopancreas and midgut, the sites where this virus is detected by standard histological methods. The probes did not react to shrimp infected with Baculovirus penaei (BP = PvSNPV).

Animals↗

PubMed1990

The use of DNA probes for confirming enterotoxin production by Staphylococcus aureus and micrococci.

DNA-DNA colony hybridization was employed to evaluate the results obtained by different immunological methods for detection of staphylococcal enterotoxin. Staphylococcus aureus strains tested for staphylococcal enterotoxin production by immuno-assays and micrococci not previously tested for staphylococcal enterotoxin production were examined for presence of the genes encoding for staphylococcal enterotoxin A, B, C and E by using three corresponding DNA probes. The staphylococcal enterotoxin A probe also detected staphylococcal enterotoxin E gene because of 100% homology. The optimal sensitivity plate method showed the best accordance between the immuno-assay and the hybridization reactions. The enzyme-linked immunosorbent assay detected 12.5 to 17% staphylococcal enterotoxin producers without hybridization reactions. The microslide gel double diffusion test and the reversed passive latex agglutination test showed rather poor accordance with the hybridization reactions. All 17 strains of different micrococci investigated were negative in hybridization with all three DNA probes.

Base Sequence↗

PubMed1990

Comparison of a DNA probe, complement-fixation and indirect immunofluorescence tests for diagnosing Anaplasma marginale in suspected carrier cattle.

Most estimates of the prevalence of anaplasmosis have been based on serologic data using the complement-fixation (CF) and/or card agglutination tests. Since these tests are considered to be only about 50 percent reliable for detecting carrier cattle in enzootically stable herds, the need for more sensitive diagnostic tests is widely recognized. The objective in the present study was to compare the sensitivity of the CF test with that of the indirect immunofluorescence (IIF) test and a recently developed DNA probe in determining the prevalence of Anaplasma marginale infection in cattle from an enzootic area. The study herd consisted of 52 8-month-old steers and 13 3-year-old cows of mixed beef breed. All cattle were initially tested for this comparative purpose. All but one animal (one that was a positive reactor as assessed by all three tests, and served as a positive control), were treated with long-acting oxytetracycline in an attempt to clear any carrier infections. Each animal was then retested at 1 month and 2 months post-treatment (PT), in an effort to determine if the DNA probe could be used to evaluate the effectiveness of the drug. Six of the 65 (9.2%) initial serum samples were CF positive. In contrast, 60 (92.3%) and 64 (98.5%) of the samples were positive as assessed with the IIF test and the DNA probe, respectively. The DNA hybridization reactions varied in intensity within the sample population indicating different individual levels of infection. The DNA probe hybridized with two samples taken at 1 month PT, and with two different samples taken at 2 months PT. The mean IIF titers were reduced at both the 1 month and 2 month sampling times. These results suggest that the drug did not eliminate infections in all cattle. Some may have been cleared, but, in any event, the drug did reduce the level of infections below the sensitivity of the DNA probe and interrupted continuity of stimulation of antibody. Therefore, the DNA probe and the IIF test appear to be considerably more sensitive in detecting carrier infections than the CF test, and should be considered in future epidemiologic studies.

Anaplasma↗

PubMed1993

Diagnostic efficacy of a DNA probe in pneumonia caused by Legionella species.

A commercial DNA probe kit (Gen-probe) for the detection of rRNA from legionellae was evaluated for its accuracy in diagnosing Legionnaires' disease in 167 patients with pneumonia. The test was performed on freshly obtained clinical respiratory tract samples. Cultures and direct immunofluorescence antibody (DFA) staining of the samples and serological tests were performed simultaneously for all patients. The probe assay result was positive in six patients; five of them had other laboratory evidence of disease (positive cultures or positive serological results or both). Depending on the diagnostic criteria, the probe test had a sensitivity of 31-67%, a specificity of 99% and positive predictive values of 67-83%. The diagnostic performance of the DNA probe assay in this study was superior to that of the DFA test. The results indicate that the examination of respiratory tract secretions by the Gen-probe kit is a suitable screening test for the diagnosis of Legionnaires' disease.

DNA Probes↗

PubMed1994

A rapid method for determination of susceptibility of Mycobacterium tuberculosis to isoniazid, using acridinium-ester-labeled DNA probe.

A rapid isoniazid (INH) susceptibility test was developed for Mycobacterium tuberculosis by using acridinium-ester (AE)-labeled DNA probe. The method was applied to two reference strains and 20 clinical isolates, then compared with the standard proportion method. By comparing the difference of log relative light units (RLUs) M. tuberculosis cultures incubating in the presence and absence of INH, INH susceptibility could be determined with the AE-DNA probe after three to five days of incubation. The difference of log RLUs of susceptible strains was statistically significantly lower than that for resistant strains after incubation for three to five days. The cutoff value was defined as "mean + one standard deviation" of the difference between log RLU(INH) and log RLU(no INH) on Day 5. By this criterion, agreement between the AE-DNA probe and the proportion concentration method was found in 19 of 20 susceptibility tests (95%). The AE-DNA probe test is rapid and non-isotopic, and may provide a useful alternative method for INH susceptibility testing for clinical isolates of M. tuberculosis.

Acridines↗

PubMed1991

[Analysis of genetic distances between populations using human DNA "fingerprints" detected by a phage M13 DNA probe].

The frequencies of different electrophoretic bands in DNA fingerprints detected by phage M13 DNA probe in two populations of the Kirov district were determined. The DNA polymorphisms observed in these two populations were compared with each other and with those of the Krasnodar populations, and pseudogenetic distances were calculated. The mean genetic distance between two Kirov populations was 0.072, this between every Kirov and Krasnodar population being 0.21 and 0.22.

Bacteriophages↗

PubMed1992

A species-specific DNA probe for Providencia stuartii identification.

A DNA probe is described that can be used for identification of Providencia stuartii by means of filter hybridization assays. The probe, which is a fragment of the P. stuartii phoN gene coding for an acid phosphatase, appeared to be able to recognize only P. stuartii strains in slot-blot hybridization experiments performed with total DNA extracted from 545 strains of 64 different Gram-negative bacterial species, including all the major representatives of the family Enterobacteriaceae. Owing to the problems that may be often encountered for correct identification of P. stuartii at the species level when using commercial identification systems, this probe may result useful for fast and reliable identification of P. stuartii strains for taxonomical, epidemiological and diagnostic studies.

Acid Phosphatase↗

PubMed1995

Detection of Y-bearing porcine spermatozoa by in situ hybridization using digoxigenin-labeled, porcine male-specific DNA probe produced by polymerase chain reaction.

This study was carried out to determine whether Y-bearing porcine spermatozoa could be detected by in situ hybridization using a digoxigenin (Dig)-labelled DNA probe specific to the Y chromosome produced by polymerase chain reaction (PCR). A conventional PCR (with Dig-dUTP) was performed using a set of oligonucleotide primers (5'-AAGTGGTCAGCGTGTCCATA-3' and 5'-TTTCTCCTGTATCCTCCTGC-3') for 236 bp fragment of porcine male-specific DNA sequence and 1.25 x 10(4) template white blood cells obtained from a boar. When fluorescence in situ hybridization with the Dig-labelled DNA probe was applied to the metaphase chromosome spreads prepared from both boar and gilts, the fluorescein signal was only detected on the long arm of the Y chromosome. In addition, immunocytochemical detection with the Dig-labelled DNA probe and alkaline phosphatase-labeled anti-Dig was applied to both sperm nuclei pretreated with dithiothreitol and white blood cells; 51% of sperm nuclei and 96% of white blood cells obtained from boar were labelled, whereas none of white blood cells obtained from gilts were labelled with the Dig-labelled DNA probe. The results indicated that in situ hybridization with porcine male-specific DNA probe produced by PCR made possible the direct visualization of Y-bearing porcine spermatozoa by in situ hybridization.

Animals↗

PubMed1988

Detection of cytomegalovirus in shell vial cultures by using a DNA probe and early nuclear antigen monoclonal antibody.

An in situ biotinylated DNA probe assay was evaluated as an adjunct to anti-cytomegalovirus early nuclear antigen indirect immunofluorescence and cytopathic effect on cytomegalovirus-infected monolayers in shell vial cultures. Viral infection was detected by early nuclear antigen indirect immunofluorescence at 24 h and by DNA probe assay and shell vial cytopathic effect at 5 days.

Antibodies, Monoclonal↗

PubMed1992

Comparative evaluation of a chemiluminescent DNA probe and an exoantigen test for rapid identification of Histoplasma capsulatum.

A chemiluminescent DNA probe (Accuprobe) assay developed by Gen Probe, Inc., for the rapid identification of Histoplasma capsulatum was evaluated and compared with the exoantigen test by using 162 coded cultures including Histoplasma capsulatum var. capsulatum, Histoplasma capsulatum var. duboisii, Histoplasma capsulatum var. farciminosum, Blastomyces dermatitidis, Coccidioides immitis, Paracoccidioides brasiliensis, and morphologically related saprobic fungi. Each test uses a chemiluminescent, acridinium ester-labeled, single-stranded DNA probe that is complementary to the rRNA of the target organism. Lysates of the test cultures were prepared by sonication with glass beads and heat treated. After the rRNA was released from the target organism, the labeled DNA probe combined with the target H. capsulatum rRNA to form a stable DNA-RNA hybrid. A hybridization protection assay was used, and the chemiluminescence of hybrids was measured initially with a Leader 1 luminometer as relative light units and later during the investigation with a probe assay luminometer as probe light units. Of the 162 coded mycelial cultures tested by the Accuprobe assay, 105 were identified as H. capsulatum. The test could be performed with an inoculum of a few square millimeters (1 to 2 mm2) of growth. In the primary evaluation, the Accuprobe identified 103 of the 105 cultures as H. capsulatum within 2 h. The remaining two cultures, contaminated with bacteria, had to be purified before the Accuprobe assay identified them correctly as H. capsulatum. Since each coded culture was concurrently tested for H. capsulatum, B. dermatitidis, and C. immitis exoantigens, the identification of all three dimorphic pathogens was provided simultaneously. Of the 162 coded cultures tested, 105 were identified by the exoantigen test as H. capsulatum, 12 were identified as B. dermatitidis, 13 were identified as C. immitis, and 32 were negative for H. capsulatum, B. dermatitidis, and C. immitis. The bacterial contamination in two isolates did not interfere with the exoantigen testing. The exoantigen test required 7- to 10-day-old colonies and required 48 to 72 h of incubation before definitive identification was obtained.

Antigens, Fungal↗

PubMed2004

Development of a DNA probe and a PCR based diagnostic assay for Rhizoctonia solani using a repetitive DNA sequence cloned from a Sri Lankan isolate.

Rhizoctonia solani is a destructive fungal pathogen of many economically important plants all over the world and the causative organism of sheath blight of rice in many tropical countries including Sri Lanka. A repetitive sequence from the genome of R. solani was cloned and characterized with a view to develop a DNA probe and a PCR diagnostic assay for detection of the fungus. The cloned sequence was 1550 bp long and appeared to be interspersed throughout the genome. The cloned sequence hybridized only to R. solani DNA and was sensitive enough to detect 100 pg of R. solani genomic DNA. PCR primers were designed from the cloned sequence and it was possible to develop a PCR assay for the specific detection of the fungal DNA with 10 pg sensitivity.

DNA Probes↗

PubMed1988

Detection of Chlamydia trachomatis in culture and urogenital smears by in situ DNA hybridization using a biotinylated DNA probe.

The detection of Chlamydia trachomatis by in situ DNA hybridization in urogenital smears was investigated using a commercially available biotinylated DNA probe. Intracellular staining of inclusion bodies was used as the criterion for positivity. Of 35 patients with a culture proven chlamydial infection 19 had smears in which C. trachomatis was detected by in situ DNA hybridization, indicating a sensitivity of 54%. Of 57 patients with a negative culture, two had positive smears by in situ DNA hybridization. To compare whether the criterion for positivity had influenced the sensitivity obtained with in situ DNA hybridization, 14 duplicate smears from culture positive patients were analysed with in situ DNA hybridization and immunofluorescence. Both methods detected intracellular inclusion bodies in seven of these smears, suggesting that the presence of infected cells mainly determines the sensitivity. The DNA probe did not cross-react with micro-organisms commonly found in the urogenital tract.

Biotin↗

PubMed1995

DNA probe and polymerase chain reaction procedure for the specific detection of Serpulina hyodysenteriae.

Serpulina (Treponema) hyodysenteriae, a Gram-negative anaerobic spirochete, is the causative agent of swine dysentery, a mucohaemorrhagic diarrheal disease in which lesions are confined to the large intestine of pigs. A DNA probe and polymerase chain reaction (PCR) amplification procedures which are specific, rapid , and sensitive for the detection of S.hyodysenteriae have been developed. Clone pF12 from a plasmid library of S.hyodysenteriae B204 genomic DNA was identified as a clone specific for S.hyodysenteriae but not for S.innocens by differential hybridization screening with S.hyodysenteriae and S.innocens genomic DNA probes. A DNA probe consisting of a 1.3 kb restriction fragment from pF12 was found to be highly specific for S. hyodysenteriae and detected 10(5) bacterial cells. A PCR procedure using primers derived from this fragment yielded a single product which was specifically generated for S.hyodysenteriae template DNA and not for other control cells DNA. PCR provided increased sensitivity with the direct detection of as few as 10 S.hyrodysenteriae cells. The PCR procedure could detect S.hyodysenteriae cells in seeded faecal matter. Moreover the PCR assay was able to detect most S. hyodysenteriae field isolates of serotypes 8 and 9. These tools have diagnostic application in veterinary microbiology.

Animals↗