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At least 469 records · Page 26Linked to original sources
PubMed1990

Direct detection of beta thalassemic mutations: use of biotin-labelled allele specific probes.

Mutations at positions beta IVS1-6, beta IVS1-110, and beta 39 of the beta globin gene are responsible for the three most common thalassemic genes in the Mediterranean population. The polymerase chain reaction (PCR) was employed to amplify a 536 base pair segment surrounding this region. Nonradioactive labelling of an oligonucleotide probe, specific for the beta IVS1-110 mutation, was achieved by incorporation of biotin-16-dUTP into a standard 3'-end labelling procedure. This probe was subsequently hybridized with the PCR amplification product and permitted detection of the mutant gene in a homozygous beta thalassemic child by a simple colour detection method using a streptavidin-alkaline phosphatase conjugate and NBT/BCIP (nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate) substrate. A known cloned mutant gene was similarly detected. Results could be obtained within 48 hr. These findings suggest that such an approach could provide a rapid and specific means for detection of beta thalassemic mutations without the need for radioactive probes.

Alleles↗
PubMed1990

Polymerase chain reaction amplification applied to the direct detection of a 4 bp deletion in the promoter region of the A gamma gene.

We report here the identification of a 4 bp deletion in the A gamma T globin gene promoter by means of Fnu4HI digestion of DNA amplified by polymerase chain reaction (PCR). This deletion has been previously associated with haplotype II beta-thalassemia in Sardinia. This simple, non-radioactive procedure should facilitate the screening of various populations of normal and beta-thalassemic subjects for this specific genetic alteration.

Chromosome Deletion↗
PubMed1990

A simple and rapid procedure for the direct detection of cytomegalovirus in urine samples.

We compared cytomegalovirus (CMV) isolation in tissue culture and viral DNA detection by DNA:DNA hybridization in 60 clinical urine samples concentrated by different procedures. Our results showed that urine concentration by filtration is the easiest and quickest procedure allowing further detection of CMV. With optimized working conditions, the CMV detection in urine can be done in 5 hr without requiring cell cultures or costly instruments.

Culture Techniques↗
PubMed1981

Direct detection of two cross-reactive antigens between porcine and rabbit zonae pellucidae by radioimmunoassay and immunoelectrophoresis.

Zonae pellucidae were isolated from oocytes obtained from rabbit and porcine ovaries. Antisera were prepared in rabbits against heat-solubilized porcine zonae and in guinea pigs against intact and heat-solubilized rabbit zonae and against purified rabbit zona proteins isolated from two-dimensional electrophoresis gels. These antisera were used in radioimmunoassay studies to show cross-reactivity between porcine and rabbit zona antigens. Immunoelectrophoresis studies also demonstrated the immunochemical cross-reactivity between two distinct antigens of rabbit and porcine zonae. These two rabbit zona antigens were demonstrated to be immunochemically identical to porcine zona antigens by crossed-line immunoelectrophoresis.

Animals↗
PubMed1995

Direct detection of hepatitis C virus (HCV) RNA from whole blood, and comparison with HCV RNA in plasma and peripheral blood mononuclear cells.

Hepatitis C virus (HCV) requires reverse transcriptase-polymerase chain reaction (RT-PCR) or branched DNA signal amplification assays to be detected in patient samples. Although conventional methods of RNA isolation are employed for samples of serum, plasma, and peripheral blood mononuclear cells (PBMCs), whole blood is generally considered an unsuitable source of RNA because of abundant RNases and polymerase inhibitors. Using a cationic surfactant, Catrimox-14, we adapted a procedure for RNA isolation from whole blood, plasma, and PBMCs that yields RNA template suitable for HCV RT-PCR. RNA isolation required less than 2 hr, and HCV sequences were easily detected in sample volumes of 50 microliters whole blood or plasma, and in less than 1 x 10(4) PBMC. Following the addition of blood to Catrimox, HCV RNA was stable in the mixture when incubated for at least 7 days at room temperature prior to RNA extraction. Comparison of whole blood HCV RNA and plasma HCV RNA from individuals with chronic hepatitis suggests that HDV RNA can be more reliably detected in whole blood. Three of 15 HCV antibody positive patients (20%) had HCV RNA present in whole blood but simultaneously obtained plasma samples were negative. Two of the HCV antibody negative individuals with chronic hepatitis contained HCV RNA in whole blood, yet one of these patient's plasma was negative for viral RNA. The Catrimox-14 method of RNA purification is useful for detecting HCV RNA in whole blood and blood subfractions, and provides a practical method of measuring plasma and PBMC HCV RNA from clinical specimens.

Base Sequence↗
PubMed2001

Direct detection of polyaromatic hydrocarbons, estrogenic compounds and pesticides in water using desorption chemical ionisation membrane inlet mass spectrometry.

This paper describes the use of desorption chemical ionisation membrane inlet mass spectrometry (DCI-MIMS) for the detection of a broad range of common contaminants in water. In addition, we discuss the advantages/disadvantages of two different types of chemical ionisation reagent gases, i-butene (a Broensted acid reagent) and argon (a charge exchange reagent). We found that polyaromatic hydrocarbons was detectable at ppt levels, the estrogenic compounds diethyl phthalate and octylphenol at high ppt levels, steroid hormones at ppb levels, hydrophobic pesticides at low ppb levels, whereas hydrophilic pesticides and bisphenol A were not detectable at all. With the exception of the polyaromatic hydrocarbons and pentachlorophenol, none of the reported compounds have to our knowledge been detected previously by other MIMS systems. In most cases the Broensted acid reagent gave characteristic ions at high mass/charge ratio, whereas the charge exchange reagent gave less characteristic ions at low m/z ratio. However, the sensitivity was generally not as good with the Broensted acid reagent as with the charge exchange reagent, since the Broensted acid reagent, i-butene, gave a large chemical background.

Estrogens↗
PubMed2000

A spectrophotometric method for the direct detection and quantitation of nitric oxide, nitrite, and nitrate in cell culture media.

A method for the spectrophotometric determination of nitric oxide, nitrite, and nitrate in tissue culture media is presented. The method is based on the nitric oxide-mediated nitrosative modification of sulfanilic acid that reacts with N-(1-naphthyl)ethylenediamine dihydrochloride forming an orange-colored product absorbing at 496 nm. Nitric oxide levels were determined in culture media from this absorbance measurement using chemiluminescence standardization. Extinction coefficients of 5400 and 6600 M(-1) cm(-1) were determined for the nitric oxide product in assay solutions containing 0.1 or 100 mM KPO4 buffer (pH 7.4), respectively, with a limit of detection of 1 microM. Acidification of these reactions (pH 2.4) generated a pink-colored product absorbing at 540 nm allowing for quantitation of total nitric oxide/nitrite levels using extinction coefficients of 38,000 and 36,900 M(-1) cm(-1), for the assay solutions described. The limit of detection of this assay was approximately 300 nM. Using the 100 mM KPO4 buffer system, nitrate levels were determined following reduction to nitrite using a copper-coated cadmium reagent with an extinction coefficient of 29,500 M(-1) cm(-1) and a detection limit of 0.5 microM. The utility of these assays was demonstrated in the standardization of nitric oxide-saturated cell culture media, and the release of nitric oxide by the NONOate compound DEA/NO.

Culture Media↗
PubMed2001

Aptamer beacons for the direct detection of proteins.

We have designed a new class of molecules, which we term aptamer beacons, for detecting a wide range of ligands. Similar to molecular beacons, aptamer beacons can adopt two or more conformations, one of which allows ligand binding. A fluorescence-quenching pair is used to report changes in conformation induced by ligand binding. An anti-thrombin aptamer was engineered into an aptamer beacon by adding nucleotides to the 5'-end which are complementary to nucleotides at the 3'-end of the aptamer. In the absence of thrombin, the added nucleotides will form a duplex with the 3'-end, forcing the aptamer beacon into a stem-loop structure. In the presence of thrombin, the aptamer beacon forms the ligand-binding structure. This conformational change causes a change in the distance between a fluorophore attached to the 5'-end and a quencher attached to the 3'-end. Aptamer beacon can be a sensitive tool for detecting proteins and other chemical compounds.

Circular Dichroism↗
PubMed1986

Evaluation of the direct detection of group A beta-hemolytic streptococcal antigen in a pediatric population: comparison with the traditional culture technique.

An evaluation of the Directigen Group A Strep Test (DGAST) in comparison with the traditional culture technique, was carried out on 1907 throat specimens, obtained from pediatric patients suspected of having a group A beta-hemolytic streptococcal pharyngitis. Of the 344 specimens positive by culture, 277 were DGAST positive (sensitivity, 81%). Of the 1563 specimens negative by culture, 1511 were DGAST negative (specificity, 97%). Nineteen isolates of non-group A beta-hemolytic streptococci were recovered, primarily group C, B and G. The DGAST is easy to perform, rapid and very specific, but lower sensitivity indicates that a back up traditional culture is still necessary, especially in pediatric patients.

Adolescent↗
PubMed1995

Limited value of cerebrospinal fluid for direct detection of Toxoplasma gondii in toxoplasmic encephalitis associated with AIDS.

The diagnosis of acquired immunodeficiency syndrome-associated toxoplasmic encephalitis (TE), a typically focal disease resulting from reactivation of tissue cysts, relies mainly on indirect diagnostic methods. In a prospective study, we investigated the value of detection of Toxoplasma gondii in cerebrospinal fluid (CSF) by using the polymerase chain reaction and the mouse inoculation test. Twenty-four patients with 26 episodes of TE, 2 HIV-infected patients with primary acute Toxoplasma infection, and 38 HIV-infected control patients with latent Toxoplasma infection were investigated. Detection of T. gondii in CSF by both methods was possible in only 3 of the TE patients (11.5%), the remaining patients being negative with either of the methods. In contrast, T. gondii DNA was detected in both of the acutely infected patients, indicating that in primary acute toxoplasmosis parasites may easily be found in the CSF, whereas in the majority of TE cases in immunocompromised patients, T. gondii parasites do not gain access to the CSF drawn by lumbar puncture.

AIDS-Related Opportunistic Infections↗