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[Direct detection of toxoplasma in the aqueous humor by gene amplification].

For immunocompetent patients, ocular toxoplasmosis is the most frequent infectious cause of chorioretinal inflammation. Nowadays, the laboratory diagnosis of ocular toxoplasmosis requires serological tests and anterior chamber puncture to detect the local production of specific antibodies. The authors describe a new technique to detect toxoplasma in aqueous humor by a polymerase chain reaction in which the target is a 88 bp specific rDNA fragment. 31 patients were concerned (23 highly suspect of ocular toxoplasmosis and 8 controls). The presence of the parasite in aqueous humor was found in 7 cases. No false positive was detected. The sensitivity of the test is reduced by the poor volume of the sample. The combination of this technique with Desmont's coefficient gives a better positive predictive value. We emphasize the pathophysiological value of this technique by suggesting the presence of tachizoites in the anterior chamber. According to our knowledge, this finding has never been demonstrated. In the future, this should be a very promising technique for the diagnosis of ocular toxoplasmosis.

Adolescent↗

[Evaluation of a rapid test for direct detection of group A streptococcal antigen in throat swabs. Study of 4 commercial test systems].

Four commercially prepared test reagent kits for the rapid screening of throat swabs for group A streptococci were examined in comparison to bacteriological cultures of 963 double throat swabs. The sensitivities of the test systems were 86.7% (Abbott Test Pack Strep A), 81.8% (Hybritech Tandem Icon Strep A), 79.6% (Pharmacia Phadirect Strep A), and 72.6% (Wellcome Reveal Colour Strep A) respectively. The specificity of the four tests was above 90%. Marked variations between different test reagent batches could be observed. The two tests based on the enzyme immunoassay principle proved to be superior to the agglutination tests investigated with respect to sensitivity and reading accuracy. Our results are evaluated in the light of the published literature on these tests.

Antigens, Bacterial↗

Direct detection of a missense mutation causing severe hemophilia A by PCR amplification and fluorescence scanning.

The amplification of Factor VIII gene-specific sequences, obtained by polymerase chain reaction, was used for hemophilia A carrier detection. Exon 24 sequences were employed in the carrier status determination of a missense mutation causing severe hemophilia A in two unrelated patients. After agarose gel electrophoresis, the digested DNA was subjected to quantitative determination of fluorescence. This technique significantly improves the digest analysis.

Base Sequence↗

Direct detection of a dioxygen adduct of cytochrome a3 in the mixed valence cytochrome oxidase/dioxygen reaction.

Time-resolved resonance Raman spectra have been recorded during the reaction of mixed valence (a3+ a2+(3)) cytochrome oxidase with dioxygen at room temperature. In the spectrum recorded at 10 microseconds subsequent to carbon monoxide photolysis, a mode is observed at 572 cm-1 that shifts to 548 cm-1 when the experiment is repeated with 18O2. The appearance of this mode is dependent upon the laser intensity used and disappears at higher incident energies. The high frequency data in conjunction with the mid-frequency data allow us to assign the 572 cm-1 mode to the Fe-O stretching vibration of the low-spin O2 adduct that forms in the mixed valence cytochrome oxidase/dioxygen reaction. The 572 cm-1 v(Fe2(+)-O2) frequency in the mixed valence enzyme/O2 adduct is essentially identical to the 571 cm-1 frequency we measured for this mode during the reduction of O2 by the fully reduced enzyme (Varotsis, C., Woodruff, W. H., and Babcock, G. T. (1989) J. Am. Chem. Soc. 111, 6439-6440; Varotsis, C., Woodruff, W. H., and Babcock, G. T. (1990) J. Am. Chem. Soc. 112, 1297), which indicates that the O2-bound cytochrome a3 site is independent of the redox state of the cytochrome a/CuA pair. The photolabile oxy intermediate is replaced by photostable low- or intermediate-spin cytochrome a3+(3), with t1/2 congruent to 200 microseconds.

Animals↗

[Direct detection of Actinobacillus (Haemophilus) pleuropneumoniae in the lungs of swine].

By means of cultural examination, coagglutination test (CT) and indirect fluorescent-antibody-technique (IFAT) a total of 199 lung specimens from necropsy pigs from Northwestern Germany with symptoms of pleuropneumonia was examined for Actinobacillus (Haemophilus) pleuropneumoniae (AP). The CT was used to detect type specific antigens in lung extracts and the IFAT was performed on tissue sections. Both tests were found to be specific. Detection and identification of AP by either test were successful in 68 of 199 lung specimens. AP was isolated out of 40 lungs, antigen detection by CT was successful in 40 and by IFAT in 65 lung samples. In 26.5% of the positive samples AP was demonstrated only by IFAT. In 4.4% of the positive specimens AP was demonstrated only by cultural examination, but the detected serovars were not accounted in IFAT and CT. In 44.1% of the positive specimens AP was isolated or detected by all three techniques. The predominating serovar was serovar 9 followed by 2 and 7. One field isolate could be identified as serovar 3 and another one as serovar 10. Furthermore one isolate was untypable. IFAT and CT were limited for detection of serovars 2, 7 and 9. Detection of multiple serovars in few lung samples was successful only by IFAT. Indirect fluorescent-antibody-technique was found to be more sensitive than coagglutination test and cultural examination. On the other hand CT was found to be less time consuming and easier to evaluate than other tests. By this, coagglutination test seems to be preferable in examining large numbers of lung samples.

Actinobacillus↗

Direct detection of usual and atypical alleles on the human aldehyde dehydrogenase-2 (ALDH2) locus.

A method for determining human mitochondrial aldehyde dehydrogenase (ALDH2) genotypes was developed. Two 21-base synthetic oligonucleotides, one complementary to the usual ALDH2(1) gene and the other complementary to the atypical ALDH2(2) gene, were used as specific probes for in-gel hybridization analysis of human genomic DNA from either peripheral blood cells or livers. Under appropriate hybridization conditions, these two probes can hybridize to their specific complementary alleles and thus allow the genotyping of the ALDH2 locus.

Aldehyde Dehydrogenase↗

Direct detection of Chlamydia trachomatis in clinical samples.

Some properties of a monoclonal antibody raised against the major outer membrane protein of Chlamydia trachomatis strain L2 are described. The antibody was fluorescein (FITC) conjugated and was provided as a diagnostic kit (Chlamyset Antigen Test, Orion Diagnostica). The monoclonal antibody reacted well with strains K, L1, L2, D, E, and G, and moderately well with the strains F, C, J, A, H and I of C. trachomatis. It did not react with the human C. psittaci strain Tw 183. The reagent had a sensitivity of 89.3% and a specificity of 98.7% when compared to parallel isolation in tissue culture. The monoclonal reagent seemed to be sensitive and very specific for rapid detection of Chlamydia trachomatis in clinical samples.

Antibodies, Monoclonal↗