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PubMed2001

Comparison of Chemicon SimulFluor direct fluorescent antibody staining with cell culture and shell vial direct immunoperoxidase staining for detection of herpes simplex virus and with cytospin direct immunofluorescence staining for detection of varicella-zoster virus.

A new rapid direct immunofluorescence assay, the SimulFluor direct fluorescent-antibody (DFA) assay, which can simultaneously detect herpes simplex virus types 1 and 2 (HSV-1 and -2) and varicella-zoster virus (VZV), was evaluated in comparison with our current standard procedures of (i) shell vial direct immunoperoxidase (shell vial IP) staining and cell culture for detection of HSV and (ii) cytospin DFA staining for VZV detection. A total of 517 vesicular, oral, genital, and skin lesion specimens were tested by all three procedures. For HSV detection, the SimulFluor DFA assay had an overall sensitivity, specificity, positive predictive value, and negative predictive value of 80.0, 98.3, 92.3, and 95.1%, respectively, when compared to culture. Shell vial IP staining had a sensitivity, specificity, positive predictive value, and negative predictive value of 87.6, 100, 100, and 96.9%, respectively, when compared with cell culture. The SimulFluor DFA assay, however, offers same-day, 1.5-hours results versus a 1- to 2-day wait for shell vial IP staining results and a 1- to 6-day wait for culture results for HSV. For VZV detection SimulFluor DFA staining detected 27 positive specimens as compared to 31 by our standard cytospin DFA technique--a correlation of 87.1%. A positive SimulFluor reaction for VZV is indicated by yellow-gold fluorescence compared to the bright apple-green fluorescence observed by cytospin DFA staining. There is no difference in turnaround time between the two assays. The SimulFluor DFA assay is a rapid immunofluorescence assay that can detect 80% of the HSV-positive specimens and 87% of the VZV-positive specimens with a 1.5-h turnaround time.

Animals↗

PubMed2000

A rapid DNA isolation procedure for the identification of Campylobacter jejuni by the polymerase chain reaction.

We have developed an efficient process for rapidly isolating campylobacter DNA using mechanical disruption combined with the guanidine-based reagent DNAzol. Template DNA was isolated by this method from cultures of Campylobacter jejuni resistant to lysis by boiling or enzymes and identified following polymerase chain reaction (PCR) amplification using primers specific for the hippuricase gene. Direct detection of campylobacters in poultry-processing samples by PCR is demonstrated in chicken carcass rinses spiked with lysis-resistant C. jejuni. Our results indicate that this method of DNA isolation may be ideal for direct PCR detection of pathogenic bacteria in complex samples of widely varied origin, especially when the target organisms are difficult to lyse by other means.

Animals↗

PubMed1998

B19 parvovirus induced fetal hydrops: rapid and simple diagnosis by detection of B19 antigens in amniotic fluids.

In our study we describe the direct detection of parvovirus B19 capsid antigens in amniotic fluid samples for the rapid and simple prenatal diagnosis of B19 induced fetal hydrops. The assay was performed on amniotic fluid specimens from fetal hydrops dotted on nylon membranes. The two capsid antigens, VP1 and VP2, which represent four per cent and 96 per cent of the capsid, respectively, were detected using a pool of monoclonal antibodies directed against these two proteins and the complex was visualized by immunoperoxidase staining. The assay could be performed in about four hours and positive results were revealed at the end of the reaction as dark blue spots on the nylon membrane. We analysed 26 amniotic fluid samples from 26 selected cases of non-immune hydrops for the presence of B19 antigens. Out of these 26 samples, 13 had previously proved positive for B19 DNA, detected by dot blot hybridization and/or in situ hybridization and/or nested PCR, and 13 had proved negative. The results obtained with our assay were compared with results obtained for the presence of B19 DNA and a close agreement was found. The method is simple and rapid to perform, does not require costly instruments, and all the reagents used in the assay are commercially available. The assay described can thus be useful for a prompt counselling and management of B19 fetal infection.

Amniotic Fluid↗

PubMed1992

Polymerase chain reaction for the detection of Neisseria gonorrhoeae in clinical samples.

AIMS: To evaluate the use of a cppB gene derived polymerase chain reaction (PCR) assay for direct detection of Neisseria gonorrhoeae in clinical samples. METHODS: A PCR assay was performed on 33 N gonorrhoeae strains and 12 other Neisseria species and other normal genital flora to evaluate the specificity of the chosen cppB primers. The assay was subsequently evaluated with 52 clinical swab samples collected from China. RESULTS: An amplified product of 390 base pairs (bp) was observed with all the N gonorrhoeae strains, each of these products on digestion with the restriction enzyme MspI produced two bands of 250 bp and 140 bp respectively. This set of primers did not produce any amplified product of the expected length with the other non-gonococcal strains tested. For the 52 clinical swabs, 34 were culture positive and PCR successfully detected all these positives. In addition the PCR was positive for two swabs which were culture negative but positive for N gonorrhoeae antigens when tested with the ELISA method (Gonozyme). CONCLUSIONS: This PCR assay is a promising diagnostic tool for detection of gonococci directly from clinical swab samples. Further evaluation is necessary.

Amino Acid Sequence↗

PubMed2001

Rapid and simple single nanogram detection of glycoproteins in polyacrylamide gels and on electroblots.

The fluorescent hydrazide, Pro-Q Emerald 300 dye, may be conjugated to glycoproteins by a periodic acid Schiff's (PAS) mechanism. The glycols present in glycoproteins are initially oxidized to aldehydes using periodic acid. The dye then reacts with the aldehydes to generate a highly fluorescent conjugate. Reduction with sodium metabisulfite or sodium borohydride is not required to stabilize the conjugate. Though glycoprotein detection may be performed on transfer membranes, direct detection in gels avoids electroblotting and glycoproteins may be visualized within 2-4 h of electrophoresis. This is substantially more rapid than PAS labeling with digoxigenin hydrazide followed by detection with an antidigoxigenin antibody conjugate of alkaline phosphatase, or PAS labeling with biotin hydrazide followed by detection with horseradish peroxidase or alkaline phosphatase conjugates of streptavidin, which require more than eight hours to complete. Pro-Q Emerald 300 dye-labeled gels and blots may be poststained with SYPRO Ruby dye, allowing sequential two-color detection of glycosylated and nonglycosylated proteins. Both fluorophores are excited with mid-range UV illumination. Pro-Q Emerald 300 dye maximally emits at 530 nm (green) while SYPRO Ruby dye maximally emits at 610 nm (red). As little as 300 pg of alpha 1-acid glycoprotein (40% carbohydrate) and 1 ng of glucose oxidase (12% carbohydrate) or avidin (7% carbohydrate) are detectable in gels after staining with Pro-Q Emerald 300 dye. Besides glycoproteins, as little as 2-4 ng of lipopolysaccharide is detectable in gels using Pro-Q Emerald 300 dye while 250-1000 ng is required for detection with conventional silver staining. Detection of glycoproteins may be achieved in sodium dodecyl sulfate-polyacrylamide gels, two-dimensional gels and on polyvinylidene difluoride membranes.

Animals↗

PubMed1987

DNA techniques in prenatal diagnosis and in genetic pathology.

New molecular techniques concerned with the isolation and identification of DNA fragments can be used for carrier detection and early prenatal diagnosis either by direct detection of the mutant DNA sequence or by indirect linkage studies employing RFLPs as DNA markers. Gene specific DNA probes are available already for a number of genetic disorders, such as the hemoglobinopathies, hemophilia A and B, alpha 1-antitrypsin deficiency, phenylketonuria, and chronic granulomatous disease. Coinheritance of DNA-polymorphisms can be traced, eg, for Norrie disease, myotonic dystrophy, Duchenne and Becker muscular dystrophies, and Huntington chorea. Several genes have been localized successfully to specific chromosome regions. By "walking" or "jumping" along the chromosome, it is hoped finally to reach further gene loci of interest, to analyze the molecular pathology of single gene disorders, and to find new ways for their prevention.

DNA↗

PubMed2003

Detection of glycoproteins in polyacrylamide gels and on electroblots using Pro-Q Emerald 488 dye, a fluorescent periodate Schiff-base stain.

Pro-Q Emerald 488 glycoprotein stain reacts with periodic acid-oxidized carbohydrate groups, generating a bright green-fluorescent signal on glycoproteins. The stain permits detection of less than 5-18 ng of glycoprotein per band, depending upon the nature and the degree of protein glycosylation, making it roughly 8-16-fold more sensitive than the standard colorimetric periodic acid-Schiff base method using acidic fuchsin dye (pararosaniline). The green-fluorescent signal from Pro-Q Emerald 488 stain may optimally be visualized using charge-coupled device/xenon arc lamp-based imaging systems or 470-488 nm laser-based gel scanners. Though glycoprotein detection may be performed on transfer membranes, direct detection in gels avoids electroblotting and the specificity of staining is better in gels. After detecting glycoproteins with Pro-Q Emerald 488 dye, total protein profiles may subsequently be detected using SYPRO Ruby protein gel stain. Using computer-assisted registration techniques, images may then be merged to generate differential display maps.

Acrylic Resins↗

PubMed1990

Preparative high-performance liquid chromatography of minor products of Streptomyces cinnamonensis.

Analytical and preparative high-performance liquid chromatography of 3 phenazines and furonaphthoquinone derivative on reversed-phase column are described. The mobile phase was methanol and water. The injected amount of the mixture was about 30 mg for a preparative chromatographic run requiring 80 min. Substances were detected directly in the column effluent by UV detection.

Chromatography, High Pressure Liquid↗

PubMed2001

Porcine endogenous retroviruses (PERVs): generation of specific antibodies, development of an immunoperoxidase assay (IPA) and inhibition by AZT.

Xenotransplantation may be associated with the risk of transmission of microorganisms. In particular, the porcine endogenous retroviruses (PERV) have raised concerns as in vitro experiments show susceptibility of human cells for PERV infection. However, it remains unclear whether PERVs are able to infect transplant recipients in vivo and whether they are pathogenic. It is therefore essential that the risks are evaluated and for this purpose specific and sensitive screening methods for PERVs have to be developed. We generated specific antibodies against all major structural proteins of PERV and developed several assays which allow antibodies against PERV to be detected as indirect evidence of infection. For direct detection of PERV production, reverse transcriptase (RT) assays were used. PCR methods were used to detect provirus integration and the presence of viral mRNA. Here we present an immunoperoxidase assay (IPA), which would allow the detection of viral proteins in infected cells as well as antibodies against PERV in the serum of an infected host. The specificity of the sera used in the assay was determined by several methods, including immunoelectron microscopy, and the sensitivity of the assay was compared with other methods. This IPA was used to detect PERV infection in in vitro experiments for evaluation of the virus host range, for titrating the virus and for testing anti-viral properties of AZT. Using this method it was shown that AZT inhibits replication of PERV. This IPA may be very useful for the surveillance of preclinical and clinical xenotransplantations.

Amino Acid Sequence↗

PubMed2001

Implications of muon anomalous magnetic moment for supersymmetric dark matter.

The anomalous magnetic moment of the muon has recently been measured to be in conflict with the standard model prediction with an excess of 2.6sigma. Taking the excess at face value as a measurement of the supersymmetric contribution, we find that at 95% confidence level it imposes an upper bound of 500 GeV on the neutralino mass and forbids Higgsinos as being the bulk of cold dark matter. Other implications for the astrophysical detection of neutralinos include an accessible minimum direct detection rate, lower bounds on the indirect detection rate of neutrinos from the Sun and the Earth, and a suppression of the intensity of gamma ray lines from neutralino annihilations in the galactic halo.

Journal Article↗

PubMed1994

Detection of Bacteroides fragilis in clinical specimens by PCR.

The direct detection of Bacteroides fragilis from clinical specimens was examined by using the PCR method for amplifying a specific fragment of the glutamine synthetase gene from B. fragilis. By this method, all five B. fragilis strains tested were detected, but DNAs from anaerobic bacteria of 24 other species tested, from aerobic bacteria of 12 species tested, and from human leukocytes were not amplified. Using the nested PCR method, we were able to detect as little as one bacterial cell or 100 fg of chromosomal DNA of B. fragilis. A total of 39 clinical specimens, which consisted of 19 bronchial aspirates, 10 percutaneous lung aspirates, 2 transtracheal aspirates, 6 pleural fluid specimens, and 2 pus specimens, were tested. All four culture-positive samples, of which two were bronchial aspirates, one was pleural fluid, and one was pus, were positive by PCR. Among 35 culture-negative samples, 2 bronchial aspirates were positive by PCR. One was from a patient whose two previous samples were positive by both culture and PCR. It had been submitted for culture several hours after collection, and clindamycin had been administered to the patient before collection of the specimen. The other bronchial aspirate positive by PCR was from a pneumonia patient who had also been administered clindamycin. We believe that B. fragilis was present in these two specimens but that either it was dead, it was below the level detectable by culture, or the process of anaerobic culture was unsuccessful. Thus, the PCR method may be considered useful for the sensitive and rapid detection of anaerobes in clinical specimens.

Bacteroides Infections↗

PubMed1999

Human mutations and their detection by gene and linkage analysis, allele sharing and association methods.

It has been 20 years since DNA analysis was first used in the detection of sickle-cell anaemia. Here, techniques for detecting human mutations are reviewed. We describe direct detection of mutations using restriction enzyme analysis and polymerase chain reaction amplification to detect gene deletions, rearrangements and point mutations. Indirect detection of mutations include the use of DNA polymorphisms in linkage analysis.

Chromosome Mapping↗

PubMed2004

[Virology, diagnostic tests, epidemiology and transmission mechanisms of hepatitis C virus infection].

The hepatitis C virus is a human virus, classified within a third type (Hepacivirus) of the Flaviviridae family. It is a spherical virus, of approximately 50 nm in diameter, with a glycoprotein covering that contains lipids, and its genome is a simple chain RNA molecule. It is characterised by a high degree of genomic heterogeneity, whose evolutionary consequence in the long term is the appearance of genetically different viral groups, genotypes and quasispecies. There are different diagnostic techniques for detecting hepatitis C virus infection. Serological assays: the detection of specific IgG against HCV by means of enzyme immunoassays is the most practical method for diagnosing infection by this virus. Supplementary immunblot tests are employed to confirm the specificity of the results of the EIA test. Molecular assays: qualitative and quantitative techniques have been developed for detecting RNA-HCV, based on the direct detection of the virions. The pathogeny of hepatitis C is not well understood. Its world prevalence is estimated at some 3%, which is why routine screening for its detection is not recommended. HCV transmission basically occurs through percutaneous exposure to infected blood, with higher rates observed in imprisoned persons, vagabonds, intravenous drug addicts, haemophiliacs and patients on haemodialysis. Although it can be transmitted sexually, it seems that this path is not very efficient, with a greater prevalence observed in persons with multiple sexual partners. Vertical transmission is estimated at some 2%, reaching 20% in cases of maternal coinfection with HIV.

Hepacivirus↗

PubMed1989

Detection of adducts arising from human exposure to N-nitroso compounds.

Humans are exposed to carcinogenic nitroso compounds (NOC), which are likely to result in the formation of DNA adducts. DNA adducts can be detected in human samples using a range of different analytical methodologies, including high pressure liquid chromatography (HPLC)-fluorescence, immunoassays and gas chromatography-mass spectrometry. Immunohistochemical studies offer the possibility of detecting adducts in single cells, but require further development for human studies. Sensitive 32P-postlabelling methods, in conjunction with HPLC separation, allow the detection of NOC-derived alkylated nucleotides in small samples of DNA derived from human tissues such as lymphocytes and placenta. In many studies, adducts have been detected in human DNA, but are often present, to a similar extent, in control and exposed subjects. In a number of studies, exposure to NOC has been inferred from the presence of characteristic alkyl adducts. In subjects from high risk areas for oesophageal cancer, DNA from target tissue contained higher levels of 0(6)-methyldeoxyguanosine than controls. The analysis of adducts in 'surrogate' DNA from peripheral lymphocytes appears promising as an accessible measure of alkylation damage. Also, the measurement of excreted levels of alkylpurines has the potential to be a noninvasive indication of short-term exposure to NOC. Endogenous synthesis of NOC can occur by a number of possible pathways in humans, and measurements of adducts will be a means of detecting the resulting alkylating agents, since their direct detection would be extremely difficult.

Alkylation↗

PubMed1997

DNA amplification methods for diagnosis and epidemiological investigations of avian mycoplasmosis.

Rapid, sensitive and specific tests that detect nucleic acid from pathogenic mycoplasmas are very attractive for the laboratory detection of infected flocks, and methods for direct detection of the four main pathogenic mycoplasmas have been developed. Moreover, most avian mycoplasma species can be differentiated, according to their unique restriction fragment length polymorphism (RFLP) patterns generated with different restriction enzymes. However, this method is limited to the identification of pure cultures of avian mycoplasmas as other bacteria may be amplified by the set of primers chosen. Another application of PCR-RFLP is the ability to distinguish between very closely related species such as M. gallisepticum and M. imitans. In order to characterise isolates below the species level, PCR-based subtyping methods have been introduced. One of them, arbitrarily primed-PCR, results in strain-specific arrays of DNA fragments that can distinguish even closely related strains of a given species. This method was successfully used to investigate the molecular epidemiology of vaccine strains or of Mycoplasma gallisepticum conjunctivitis in songbirds. Major issues in the development of DNA amplification tests concern the selection of the appropriate target for amplification, specimen collection, DNA preparation and detection of amplification reaction inhibitors. Detection of amplified products is most commonly performed after gel electrophoresis or probe-based methods. Careful consideration to the design and work flow of the facility are necessary to avoid false-positive results.

Agglutination Tests↗

PubMed1977

[Directivity a qualitative function of hearing aids (author's transl)].

"Directivity" is the name applied by the authors to the overall audioprosthetic phenomena which they have demonstrated in their research into the correction of deficient hearing, using hearing aids equipped with directional microphones. They propose a double blind test in order to attempt to demonstrate objectively the reasons which experimentally and in the Laboratory decide the choice of a hearing aid with a directional microphone or an omni-directional microphone. Amongst other things, the test makes it possible to confirm whether the improvement in intelligibility in a recreated noisy atmosphere is linked or not to the possibilities of directional detection offered by aids equipped with a directional microphone.

Adult↗

PubMed1985

Guanine nucleotide regulation of a mammalian myocardial muscarinic receptor system. Evidence for homo- and heterotropic cooperativity in ligand binding analyzed by computer-assisted curve fitting.

Highly purified dog heart sarcolemmal membranes, with a content of approximately 5 pmol of muscarinic acetylcholine receptor (mAChR)/mg of protein, were analyzed for mAChR-mediated inhibition of adenylyl cyclase and ligand binding in the absence and the presence of guanine nucleotides. Adenylyl cyclase was found to be coupled to the mAChR, being attenuated approximately 30% in a GTP-dependent manner. Direct binding studies, using 3H-labeled oxotremorine M, showed high affinity binding (apparent KD = 10 nM) that was reduced on nucleotide addition. Dose-response curves for GDP, GTP, and guanyl-5'-yl imidodiphosphate showed them to be equipotent. On the basis of pirenzepine binding, only one type of mAChR, commonly referred to as M2, was detected. Direct binding of [3H]quinuclidinyl benzilate [( 3H]QNB) uncovered 50% more binding sites than 150 nM 3H-labeled oxotremorine M; addition of guanine nucleotides uncovered the existence of positive cooperativity in the binding of [3H]QNB. Agonist displacement curves of [3H]QNB binding, without and with guanine nucleotides, extended over several orders of magnitude, which is inconsistent with single site competitive kinetics. The results and their analysis by computer-assisted curve fitting indicated that the data are well fitted by a model in which a receptor is at least bivalent and exists in two states: one with and the other without cooperativity between its sites, with guanine nucleotides decreasing both the degree of cooperativity between the sites and the proportion of the receptor that is in the cooperative form. Since the guanine nucleotide effect is mediated by the Ni coupling protein, it is suggested that direct binding detects R'Ni complexes (cooperative), R"NiG complexes (cooperative but distinct from R'Ni), and R0 complexes (non-cooperative and unaffected by Ni or NiG), where R = mAChR, Ni = the inhibitory regulatory component of adenylyl cyclase unaffected by guanine nucleotide, and NiG = Ni affected by guanine nucleotide (G).

Adenylyl Cyclases↗

PubMed2003

Direct determination of monolinuron, linuron and chlorbromuron residues in potato samples by gas chromatography with nitrogen-phosphorus detection.

A gas chromatography with nitrogen-phosphorus detection direct method for methoxyurea herbicide determination in powdered potato and fresh potato samples has been developed. A previous study of the thermal stability of the phenylurea herbicides seems to confirm that the ones containing the methoxy radical, i.e. monolinuron, linuron and chlorbromuron, were stable. The herbicides were extracted from the sample through liquid-liquid extraction with dichloromethane-light petroleum (1:1), followed by solid-phase extraction in a C8 cartridge. The recoveries were in the range 84-95% for powdered potato and 86-101% for fresh potato. The RSD values were less than 10%, at 0.1 microg g(-1) concentration level (n = 4) for both types of samples. Detection limits of the method were 7.0-30 ng g(-1) for powdered potato and 6.0-50 ng g(-1) for fresh potato.

Chromatography, Gas↗