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At least 163 records · Page 9Linked to original sources

PubMed1994

Synthesis of a non-radioactive hepatitis B virus DNA probe from human serum by the polymerase chain reaction.

A method for synthesizing probes for detecting hepatitis B virus DNA in serum was developed. It uses DNA extracted from the serum of an hepatitis B virus carrier as target, and digoxigenin-11-dUTP incorporated in DNA sequences during the polymerase chain reaction as tracer. Using a spot hybridization assay, the sensitivity and specificity of the digoxigenin-labelled DNA probe were compared with two standard hepatitis B virus DNA probes, synthesized with cloned hepatitis B virus DNA and labelled either with digoxigenin or 32P by random priming. Data obtained from the three methods showed an excellent correlation. Thus, hepatitis B virus DNA extracted from human serum and labelled by polymerase chain reaction may be considered a suitable alternative to cloned DNA. It provides reliable probes and eliminates the need for facilities and personnel dedicated to the manipulation of clones. These advantages will allow a wider application of hepatitis B virus DNA testing in clinical practice.

Base Sequence↗

PubMed1987

Detecting the effects of toxic agents on spermatogenesis using DNA probes.

Advances in the molecular biology of spermatogenesis suggest that DNA probes can be used to monitor the effects of toxic agents in male germ cells of mammals. Molecular hybridization analyses with DNA probes can provide a reproducible methodology capable of detecting changes ranging from massive deletions to single base pair substitutions in the genome of exposed individuals. A constantly increasing number of DNA probes that can be used to detect such alterations in human sperm DNA exist for both ubiquitously expressed proteins and for genes solely expressed in the testis. In this chapter, the currently available testicular stage-specific and/or cell type-specific DNA probes and the techniques by which they can be utilized in reproductive toxicology studies are discussed. The advantages, limitations, and future technological advances of this novel biological marker system for the human male reproductive system are also considered.

Animals↗

PubMed1992

Application of DNA probes to analysis of bacteriophage distribution patterns in the environment.

Radiolabeled bacteriophage DNA probes have been used in this study to determine the distribution of Pseudomonas aeruginosa-infecting bacteriophages in natural samples of lake water, sediment, soil, and sewage. The sensitivity of detection of bacteriophage with the DNA probes was between 10(3) and 10(4) PFU and 10(6) to 10(7) CFU of lysogenized bacteria detectable with a homologous phage DNA probe. Analyses of environmental samples suggest that up to 40% of P. aeruginosa in natural ecosystems contain DNA sequences homologous to phage genomes. By using different bacteriophage DNA probes, the diversity of the bacteriophage population in sewage was estimated to be higher than that in other natural samples. The indication that transducing phages and prophages are widely distributed in the Pseudomonas populations investigated has considerable implications for the frequency of natural gene transfer by transduction and of lysogenic conversion of host bacteria in natural ecosystems.

Bacteriophages↗

PubMed1991

Digoxygenin-labelled DNA-probe: a rapid non-radioactive method for hepatitis B virus DNA detection in serum.

The sensitivity and specificity of two non-radioactive spot hybridization assays for hepatitis B virus DNA (HBV-DNA) using biotin and digoxygenin-labelled DNA probes were investigated in parallel in 122 serum samples from patients with chronic hepatitis B and 50 controls. The results were compared with an isotopic technique using a 32P-labelled probe. HBV-DNA was detected in 56 (80%) out of 70 hepatitis B "e" antigen (HBeAg)-positive cases and in 4 (8%) out of 52 antibody to hepatitis B "e" antigen (anti-HBe)-positive cases using the digoxygenin or 32P-labelled probes. No false positives were found with either method. Using the biotin-labelled probe, 16% of sera gave discordant results, which were considered to be false positive. The time required for detection of serum HBV-DNA was 2 hours for the non-radioactive probes and 16 hours for the isotopic probes. This study suggests that the digoxygenin-labelled probe for detection of HBV-DNA is the most rapid and sensitive method for routine diagnosis of viral replication in clinical laboratories.

Alanine Transaminase↗

PubMed1996

Comparison of a DNA probe assay with the plaque reduction assay for measuring the sensitivity of herpes simplex virus and varicella-zoster virus to penciclovir and acyclovir.

A DNA probe assay was compared with the plaque reduction assay to determine the sensitivity of clinical isolates of herpes simplex virus (HSV) and varicella-zoster virus (VZV) to penciclovir and acyclovir in MRC-5 cells. In both assays, penciclovir and acyclovir shared comparable activity against cell-free virus (CFV) preparations of VZV and herpes simplex virus type 1 (HSV-1) isolates, whilst acyclovir was significantly more active than penciclovir against herpes simplex virus type 2 (HSV-2) isolates in both the DNA probe assay (P < or = 0.01) and the plaque reduction assay (P < or = 0.01). However, the 50% effective concentrations (EC50s) were generally lower in the DNA probe assay and the correlation between the plaque reduction and DNA probe assays was poor for either compound. Six acyclovir-resistant strains of HSV-1 derived in cell culture were also tested for susceptibility to penciclovir and acyclovir, in the DNA probe and plaque reduction assays. The relative susceptibilities of these strains were comparable, for example, one ACV-resistant strain was susceptible to penciclovir in both assays. Further comparisons of the assay methods were made using cell-associated VZV (CAV). As with CFV the EC50s were significantly lower in the DNA probe assay than the plaque reduction assay for penciclovir (P < or = 0.01) and acyclovir (P < or = 0.01). In the DNA probe assay there was no significant difference in the EC50s for either penciclovir or acyclovir when comparing CAV with CFV. However, in the plaque reduction assay the EC50s for CAV were significantly higher than those for CFV for both penciclovir (P < or = 0.01) and acyclovir (P < or = 0.01). Overall the DNA probe assay is objective, does not require prior titration of isolates and provides opportunities for automation. It is more suitable for sensitivity testing of large numbers of clinical isolates than the well-established plaque reduction assay.

Acyclovir↗

PubMed1991

[Identification of Mycobacterium avium and Mycobacterium intracellulare using three DNA probe tests, and their distributions in Japan].

Identification of Mycobacterium avium complex (MAC) was made using three DNA probe tests for MAC: Gen-Probe Rapid Diagnostic System for the MAC (Gen-Probe Inc., San Diego, U.S.A.), AccuProbe MAC Culture Identification or Confirmation Test (Gen-Probe Inc.); and SNAP Culture Identification Diagnostic Kit (MAC) (Syngene Inc., San Diego, U.S.A.). Various strains of MAC belonging to serovars 21 to 28 were identified by the DNA probe tests and showed the following. First, Serovar 21 and 25 belonged to M. avium and M. intracellulare, respectively. Each of them reacted with species-specific probes used in the three DNA probe tests [i.e., either M. avium-probe (in SNAP test; Probe A) or M. intracellulare-probe (in SNAP test; Probe I)]. Second, serovars 22-24 and 26-28 consisted of M. intracellulare, MAC strains that reacted with Probe X of SNAP test but lacked the reactivity with M. avium- and M. intracellulare probes of all the DNA probe tests, M. scrofulaceum that showed no reactivity with M. avium- or M. intracellulare-probe or Probe X, and M. scrofulaceum that had only the reactivity with Probe X. When the disease-associated MAC strains (35 strains), isolated in the Kanto to Kyushu areas in Japan, were identified using AccuProbe test, both the M. avium and M. intracellulare strains identified by the Gen-Probe test reacted with the MAC-probe but not with the M. tuberculosis complex (MTC)-probe.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

[DNA probe for identification of sibling species of Anopheles sinensis and Anopheles anthropophagus].

Genomic DNA libraries of Anopheles sinensis and Anopheles anthropophagus were constructed. The positive clones suitable for discrimination sibling species of An. sinensis and An. anthropophagus were screened and a clone from An. sinensis DNA library was selected and the insert DNA was used as a DNA probe to test dot blot of genomic DNA from An. sinensis and An. anthropophagus. The results showed that the DNA probe hybridized with all stages of An. sinensis DNA, but had very weak hybridization signal with An. anthropophagus DNA. The probe was very sensitive and could detect as little as 7.5ng An. sinensis which represents approximately one-150th part of the total DNA from single mosquito. The results demonstrated that the DNA probe reported here could be used to distinguish species of An.sinensis from An. anthropophagus.

Animals↗

PubMed1992

Detection of Babesia bigemina infection: use of a DNA probe--a review.

The development of a repetitive DNA probe for Babesia bigemina was reviewed. The original plasmid (p(Bbi)16) contained an insert of B. bigemina DNA of approximately 6.3 kb. This probe has been evaluated for specificity and analytical sensitivity by dot blot hybridization with isolates from Mexico, the Caribbean region and Kenya. A partial restriction map has been constructed and insert fragments have been subcloned and utilized as specific DNA probes. A comparison of 32P labelled and non-radioactive DNA probes was presented. Non-radioactive detection systems that have been used include digoxigenin dUTP incorporation, and detection by colorimetric substrate methods. Derivatives from the original DNA probe have been utilized to detect B. bigemina infection in a) experimentally inoculated cattle, b) field exposed cattle, c) infected Boophilus microplus ticks, and d) the development of a PCR amplification system.

Animals↗

PubMed1990

Human papillomavirus type 16/18 in uterine cervical adenocarcinoma in situ and adenocarcinoma. A study by in situ hybridization with biotinylated DNA probes.

The distribution of human papillomavirus (HPV) DNA type 16/18 in the paraffin sections of 11 adenocarcinomas (AC), 4 adenocarcinomas in situ (AIS), and 2 adenosquamous carcinomas (AC/SCC) of the cervix was examined by in situ hybridization (ISH) with biotinylated DNA probes (B-probes). Four AC, four AIS, and one AC/SCC were positive. These results show that B-probes are an acceptable alternative to both ISH with radiolabeled probes and Southern blot hybridization (SBH), which are difficult to apply in routine pathologic laboratories. Cervical tissues infiltrated with endometrial AC were negative for HPV DNA 16/18, and it is suggested that the presence of HPV DNA 16/18 in uterine AC may be helpful in discriminating cervical AC from endometrial AC.

Adenocarcinoma↗

PubMed1999

DNA probe for beta-hemolytic group B Streptococcus. Diagnostic accuracy in threatened preterm labor.

OBJECTIVE: To determine the diagnostic accuracy of a DNA probe for beta-hemolytic group B Streptococcus (GBS) in women with threatened preterm labor. STUDY DESIGN: Two identical vaginal/perianal samples were collected from 75 pregnant women who were being evaluated for threatened preterm labor. One sample was managed in the traditional manner, with direct plating onto blood agar followed by plating after 8 and 24 hours of LIM broth enhancement. The "gold standard" was 24 hours of LIM broth enhancement followed by blood agar plating. The second sample was placed in LIM broth, and DNA probe testing was performed after incubation for 8 and 24 hours. RESULTS: The prevalence of GBS colonization by the gold standard culture was 32%. After 8 hours of incubation in LIM broth, the DNA probe had poor sensitivity (79%); however, after 24 hours of incubation in LIM broth the DNA probe sensitivity rose to 96%. The DNA probe demonstrated only one false negative result after 24 hours of LIM broth enhancement. All DNA probe results were known 25 hours after sample collection. CONCLUSION: This DNA probe gave results nearly identical to those of standard cultures and allowed a substantial saving of time.

Adult↗

PubMed2000

A rapid DNA probe method for detection of Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans.

BACKGROUND: The objective of the present study was to develop a rapid DNA probe method for the microbiological detection of periodontitis that can be used in dental clinics. By using the DNA probe, we also investigated the correlation between the occurrence of putative periodontopathic bacteria and clinical parameters. METHODS: This rapid DNA probe method minimizes the use of a water bath for ordinary hybridization and washing in order to shorten the total reaction time. The detection process could be completed within 2 hours. In order to evaluate the clinical application of the DNA probe, subgingival plaque samples were taken from patients with periodontitis before initial therapy. After the therapy, the patients were microbiologically and clinically evaluated. RESULTS: When the DNA probe method was compared with the culture method, the agreement was 88% for Porphyromonas gingivalis and 67% for Actinobacillus actinomycetemcomitans. A statistically significant association was found between the detection of P. gingivalis and probing depth, bleeding on probing (chi2 test: P <0.001, P <0.05). A significant association was also shown between the detection of A. actinomycetemcomitans and probing depth in patients aged 35 or older (chi2 test: P <0.001). The detection rate of A. actinomycetemcomitans was highest in teenagers. At shallow periodontal pocket sites (PD < or =3 mm) in teenagers, no P. gingivalis was found, while 22% of the sites harbored A. actinomycetemcomitans. After the therapy, the frequency of detection of P. gingivalis decreased significantly only in the clinically improved sites (chi2 test: P <0.001). CONCLUSIONS: The rapid DNA probe method appears promising as an efficient tool for rapid clinical detection of periodontopathic bacteria.

Adolescent↗

PubMedDate not supplied

Use of biotinylated DNA probes in screening cells obtained from cervical swabs for human papillomavirus DNA sequences.

A nonradioactive DNA-detection procedure using biotinylated DNA probes in the screening of cells from cervical swabs for DNA sequences homologous to human papillomavirus (HPV) DNA was tested. This alternative DNA-detection method yielded results comparable to those obtained with radioisotope-labeled DNA probes in 32 cases tested. This procedure obviates the special precautions required for radioisotope materials. Accordingly, this technique can be made available to many laboratories, and conclusive evidence as to the relation of HPV infection to cervical cancer may thus be accumulated.

Biotin↗

PubMed1994

Differentiation of micronuclei in mouse bone marrow cells: a comparison between CREST staining and fluorescent in situ hybridization with centromeric and telomeric DNA probes.

Immunofluorescent staining (CREST) of kinetochore proteins and in situ hybridization (FISH) with centromeric DNA probes are able to distinguish between micronuclei (MN) containing lagging chromosomes or acentric fragments. Different frequencies of signal-positive MN induced by mitomycin C (MMC) were obtained by Miller et al. (Mutagenesis, 6, 297-302, 1991) between CREST labelling and FISH with the mouse major-gamma-satellite DNA probe (major probe). Both modes of identifying the presence of an entire chromosome in a MN are theoretically prone to misclassification. Breaks induced in pericentric heterochromatin can produce fragment-containing MN with a major signal. Alternatively, alterations of kinetochore proteins can produce CREST-negative MN containing lagging chromosomes. To improve the reliability of MN differentiation two additional DNA probes, the mouse minor satellite DNA probe (minor probe) and the telomere repeat (5'-TTAGGG-3')7, have been used and double labelling has been employed with minor/major and minor/telomere probes. At 1 mg/kg of MMC the labelling frequencies of MN with CREST and the minor probe were identical (18.5 and 19%, respectively) and the major probe showed a higher labelling rate (30.5%). Using double-labelling the difference between minor and major probe responses was confirmed (17 and 31.5%, respectively). At 5 mg/kg of MMC, CREST labelling gave the lowest (6%), the minor probe gave intermediate (10 and 11.5% after single- and double-labelling, respectively) and the major probe gave the highest signal frequencies (16.5 and 15% after single- and double-labelling, respectively). The CREST and the minor signal frequencies were not significantly different at either dose of MMC whereas the minor and the major signal frequencies were significantly different.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1998

[Use of photo-anchoring of DNA probes for fluorescent in situ hybridization].

A possibility was investigated to use photo-crosslinking DNA probes for fluorescent in situ hybridization (FISH). DNA probes were modified by incorporating photonucleotides in these, containing a photoreactive group (tetrafluorobenzazid) and capable of making covalent bonds with the examined DNA, when irradiated in 300-330 nm region. The photonucleotide was incorporated into the probe either by nick-translation, or upon elongation of the hybridized probe by the Kljonow fragment. It has been shown that the DNA probe, cross-linking to a chromosome as a result of covalent bonds, is not removed from the place of hybridization under consequent denaturating washing, which makes it possible to carry out the following DNA hybridization with selective conservation of signals obtained due to previous hybridization. This peculiarity of photo-linking DNA probes makes it possible to use them for the two-step DNA hybridization. To demonstrate this, preparations of human chromosomes were investigated. On the first step, chromosomal DNA was hybridized by means of DNA probe having nucleotide sequences of centromeric regions of chromosomes 13 and 21, the probe being linked to chromosomal DNA by the photonucleotide. Following the denaturation treatment of the preparation, and after the second chromosomal DNA hybridization with cosmid DNA, containing chromosome 13 DNA nucleotide sequence, the signal in chromosome 13 centromeric region was retained to serve a marker of this chromosome, thus fascilitating its easier identification following the hybridization of its DNA with cosmic DNA. The denaturation stability of photo-crosslinking probes opens some new possibilities in technology of DNA in situ hybridization.

Chromosomes, Human↗

PubMedDate not supplied

Comparison of DNA-probe test and culture for the detection of Neisseria gonorrhoeae in genital samples.

BACKGROUND AND OBJECTIVES: The nonisotopic DNA probe assay (PACE 2) was evaluated for the detection of Neisseria gonorrhoeae in urethral and endocervical specimens and compared quantitatively and qualitatively using recommended culture methods. GOAL OF THIS STUDY: To evaluate whether culture methods could be replaced by the DNA probe assay for the diagnosis of N. gonorrhoeae. STUDY DESIGN: A total of 502 men and women were studied, including 42 non-registered and 132 legally registered prostitutes and 115 male and 213 female STD patients. RESULTS: The overall concordance of culture and the PACE 2 assay was 98.4%. Of the tested persons, 9.6% were positive for N. gonorrhoeae by culture technique compared with 11.2% using the DNA probe assay. All samples that were positive in culture were positive in the Gen-Probe test. Of samples positive in Gen-Probe, 14.3% (8/56) were negative in culture. Three of these results were determined false-positives and five were true positives when evaluated by further analysis. The sensitivity and specificity were 90.6% and 100%, for the culture technique, and 100% and 99.3% for the DNA probe assay, respectively, when compared with true positive and negative results. CONCLUSION: The data indicate that the DNA probe assay serves as a suitable screening and diagnostic test for the diagnosis of N. gonorrhoeae in symptomatic and in asymptomatic men and women. When the performance of culture technique is not possible or unreliable, the DNA probe assay can be used for testing those at high risk for gonorrhea. In borderline cases with a low value of RLU, the DNA test should be confirmed to avoid false positive results, especially in women.

Bacteriological Techniques↗

PubMed2002

[Establishment and evaluation of the method for detecting HBV DNA in serum using HBV DNA probe labeled directly by alkaline phosphatase].

OBJECTIVE: To establish a sensitive and specific technique for detecting HBV DNA in serum using HBV DNA probe labeled directly by alkaline phosphatase (AlkPhos Direc probe). METHODS: The probe that purified HBV DNA sequence was labeled directly by alkaline phosphatase and chemiluminescent substrate CDP-star for AP was used in the hybridization assay. HBV DNA was detected by autoradiography on the film. The test compared the chemiluminescen dot blot hybridization assay for 80 samples with digoxigenin-labeled HBV DNA probe detective method. The correlation of 70 samples test results between fluorescent quantitative HBV DNA PCR method and dot blot hybridization assay by AlkPhos Direc probe was analysed. RESULTS: The sensitivity of the probe labeled directly by alkaline phosphatase was 10pg at least. The coincidence was 100% compared with digoxigenin-labeled HBV DNA probe detection. A correlation coefficient of HBV DNA quantitative results between fluorescent quantitative HBV DNA PCR (QPCR) method and dot blot hybridization assay by AlkPhos Direc probe was 0.98 (P<0.01). CONCLUSIONS: The method detecting HBV DNA in serum by HBV DNA AlkPhos Direc probe is sensitive and specific. The results between two methods with AlkPhos Direc and digoxigenin-labeled HBV DNA probe are coincident completely. The correlation of HBV DNA quantitative results between fluorescent QPCR method and dot blot hybridization assay by AlkPhos Direc probe is satisfactory.

Alkaline Phosphatase↗

PubMed1992

[DNA probe and polymerase chain reaction for detection and identification of mycobacteria].

The DNA probe and polymerase chain reaction (PCR) technique for detection and identification of mycobacteria were compared with the conventional smear and culture method. The results of identification by DNA probe agreed well with those of the biochemical method. Moreover, six percent of Mycobacterium avium complex (MAC) were revealed to be mycobacteria other than MAC by DNA probe. The nested PCR for detection of gene coding protein antigen b of Mycobacterium tuberculosis complex showed excellent specificity and sensitivity. Then we applied this technique to rapid detection of M. tuberculosis in 222 clinical samples. The agreement between nested PCR and the biochemical method was excellent, and 17 cases were diagnosed by only nested PCR in spite of negative results by smear and culture. These cases were unlikely to have yielded false positive results since their clinical features were compatible to tuberculosis. From these data, it was considered that the DNA probe and PCR technique were extremely useful strategies and would contribute to rapid diagnosis of mycobacterial infectious diseases.

Base Sequence↗

PubMed1988

Detection of DNA "fingerprints" of cultivated rice by hybridization with a human minisatellite DNA probe.

A human minisatellite DNA probe detects several restriction fragment length polymorphisms in cultivars of Asian and African rice. Certain fragments appear to be inherited in a Mendelian fashion and may represent unlinked loci. The hybridization patterns appear to be cultivar-specific and largely unchanged after the regeneration of plants from tissue culture. The results suggest that these regions of the rice genome may be used to generate cultivar-specific DNA fingerprints. The demonstration of similarity between a human minisatellite sequence and polymorphic regions in the rice genome suggests that such regions also occur in the genomes of many other plant species.

Cloning, Molecular↗