Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Direct detection”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 325 records · Page 18Linked to original sources
PubMed1979

Direct detection of methylated cytosine in DNA by use of the restriction enzyme MspI.

The extent of methylation of the internal C in the sequence CCGG in DNA from various eukaryotic sources has been determined using the restriction enzyme MspI known to be specific for this sequence. The methylation of the CCGG sequence is reflected in the restriction pattern obtained by DNA treated with MspI and its isoschizomer HpaII and analyzed by gel electrophoresis. A direct method for detection 5-methylcytosine in the sequence CCGG has been deviced. DNA fragments obtained with MspI were radioactively labeled at their 5' ends and subsequently degraded to the corresponding 5'-deoxyribonucleoside monophosphates. 5 methylcytidylic acid has been found in most of the 5' ends of MspI fragments of calf thymus DNA (about 90%) indicating heavy methylation of the sequence CCGG in calf thymus DNA. The results also reveal a symmetric methylation of both strands at this sequence in calf thymus DNA. In contrast, the CCGG sequence in other eukaryotic DNAs from organisms like Neurospora, Drosophila and Herpes virus proved to be undermethylated at this sequence.

Animals↗
PubMed1997

[Evaluation of Novapath assay for direct detection in patient stool of Vero toxin and Vero toxin-producing Escherichia coli].

We evaluated two kits which detect either Verotoxin (Novapath EHEC) or EHEC O157 (Novapath O157) directly from stool specimens and compared with the result of PCR. The tests can be completed in 3 hrs. All fecal specimens from 18 healthy volunteers showed negative results with both kits and PCR. Specimens from patients of EHEC O157 outbreak in Okayama Prefecture and asymptomatic families of patients of outbreak in Sakai City of Japan were also analyzed. Fecal specimens from which EHEC O157 was isolated showed positive results with both kits and PCR. Some specimens which were culture negative showed positive results with either Novapath EHEC or Novapath O157 or both. The sensitivity of Novapath O157 was lower than PCR to detect O157 organisms but took shorter time to get result. Novapath EHEC showed very high sensitivity in detecting Vero toxin. These results suggest that Novapath EHEC especially can be used clinically for rapid diagnosis.

Bacterial Toxins↗
PubMed1999

Direct detection and identification of Mycobacterium tuberculosis in smear-positive sputum samples by fluorescence in situ hybridization (FISH) using peptide nucleic acid (PNA) probes.

SETTING: Peptidenucleic acid (PNA) probesdesigned for specific detection of mycobacteria of the Mycobacterium tuberculosis complex (MTC) and other non-tuberculous mycobacterium species (NTM) are shown to be able to penetrate the mycobacterial cell wall and subsequently hybridize in situ to complementary rRNA. OBJECTIVE: To demonstrate the use of fluorescein-labelled PNA probes for detection and identification of M. tuberculosis in smear-positive sputum samples. DESIGN: The sensitivity and specificity of the PNA probes were investigated by fluorescence in situ hybridization (FISH) using cultures of mycobacterium strains representing species of the MTC and NTM, respectively. RESULTS: M. tuberculosis strains were detected by FISH using specific fluorescein-labelled PNA probes directly in smear-positive sputum samples without changing the morphology of the cells. CONCLUSION: PNA probes allow for rapid diagnosis of tuberculosis in smear-positive cases.

Humans↗
PubMed1989

Direct detection of point mutations by mismatch analysis: application to haemophilia B.

Rapid detection of point mutations in genomic DNA has been achieved by chemical mismatch analysis of heteroduplexes formed between amplified wild-type and target sequences in the human factor IX gene. Amplification and mismatch detection (AMD) analysis of DNA from relatives of haemophilia B patients permitted carrier diagnosis by direct identification of the presence or absence of the mutation in all cases, thus eliminating the need for the informative segregation of polymorphic markers. This extends diagnostic capability to virtually all haemophilia B families. AMD analysis permits detection of all sequence variations in genomic DNA and is therefore applicable to direct diagnosis of X-linked and autosomal diseases and for identification of new polymorphisms for genetic mapping.

Amino Acid Sequence↗
PubMed2006

Direct detection of null alleles in SNP genotyping data.

Pinpointing genetic associations in the human genome relies heavily on the accuracy of the underlying genotype data. Null alleles can generate significant inaccuracies in genotype data and can negatively affect the statistical power of a study. Existing quality control (QC) tests, including tests of Hardy-Weinberg equilibrium, are not sensitive enough to detect the presence of even moderately frequent null alleles in the data. We show that direct analysis of raw data from a quantitative genotyping platform can detect up to 75% of null alleles, even at frequencies below the sensitivity of more traditional methods. Detecting unexpected null alleles not only has benefits in QC of genotype data but may also be valuable in detecting rare, functional null alleles that would otherwise be missed.

Alleles↗
PubMed1987

Rapid commercial test for direct detection of group A streptococci in throat swabs.

The Hybritech Strep A ICON was used for direct testing of 1016 throat specimens for group A beta-hemolytic streptococci. Both the test and culture were negative in 829 specimens (81.6%); both were positive in 164 cases (16.1%); the test was positive and culture negative in 9 cases (0.9%); and the test negative and culture positive in 14 cases (1.4%).

Adult↗
PubMedDate not supplied

[Evaluation of the MTD-2 test for the direct detection of Mycobacterium tuberculosis in specimens].

BACKGROUND: To evaluate the utility of the Amplified Mycobacterium Tuberculosis Direct Test (MTD-2, Gen-Probe) in the microbiological diagnosis of tuberculosis. METHODS: We evaluated the results obtained in 146 specimens with the MTD-2 test, together with those of the culture and smears. The MTD-2 test was performed on all the smear positives specimens (n=47), on the smear-negative specimens, when the test was demanded (n=19), and in other smear-negative specimens previously selected, according to the clinical history of the patient (n=80). We considered real cases of tuberculosis, those that were culture positive for Mycobacterium tuberculosis and those that were specifically treated. RESULTS: The overall sensitivity, specificity, positive and negative predictive values for the MTD test were: 95, 76, 71, and 96%, for the culture; and 84, 100, 100 and 90% and 75, 94, 89 and 86% for the smears, respectively. In smear positive specimens, the test showed a great specificity, and differentiated M. tuberculosis from other mycobacteria. In the smear negatives, the sensitivity of the test was low and so was the positive predictive value, especially in series performed with a high work load. CONCLUSIONS: Data from our study show that the MTD-2 test is a reliable method for rapid diagnosis of tuberculosis in smear positive specimens. However, due to its low sensitivity and positive predictive value, it is not recommended in the routine diagnosis of tuberculosis. Also, for this reason, whenever a positive result is obtained with a smear negative specimen, the result needs to be confirmed with another specimen.

Bacteriological Techniques↗
PubMed2005

Contemporary mass spectrometry for the direct detection of enzyme intermediates.

The field of enzymology has long used small-molecule mass spectrometry. However, the direct interrogation of covalent and non-covalent intermediates by large-molecule mass spectrometry of enzymes or large peptide substrates is illuminating an increasingly diverse array of chemistries used in nature. Recent advances now allow improved detection of several modifications formed at sub-stoichiometric levels on the same polypeptide, and elucidation of intermediate dynamics with low millisecond temporal resolution. Highlighting recent applications in both ribosomal and non-ribosomal biosynthesis of natural products, along with acetyl transferases, sulfonucleotide reducatases, and PEP-utilizing enzymes, the utility of small- and large-molecule mass spectrometry to reveal enzyme intermediates and illuminate mechanism is described briefly. From ever more complex mixtures, mass spectrometry continues to evolve into a key technology for a larger number of today's enzymologists.

Enzymes↗
PubMed2006

Rapid analysis of organic acids in plant extracts by capillary electrophoresis with indirect UV detection: directed metabolic analyses during metal stress.

A fast, reliable capillary zone electrophoresis (CZE) method with indirect UV detection was optimized and validated to determine the main organic acids contained in plants. Citric, malic, succinic, oxalic, formic, fumaric, acetic acids, and phosphate were quantified. A rapid separation while keeping a good resolution was obtained by optimizing capillary length, separation voltage, electrolyte composition, and pH. Analyses were performed in a 30 cm uncoated fused-silica capillary (length to the detector window) in the co-electroosmotic mode with reversed electroosmotic flow and anodic detection using a -30 kV separation voltage. The pH 9.0 electrolyte contained 3 x 10(-4)mol/L tetradecyltrimethylammonium and 10(-2)mol/L trimellitate. Separation with baseline return was achieved in 100 s. Linearity, detection limits, repeatability, reproducibility, and recoveries were evaluated. Mean precision values of 0.2 and 3.4% for migration times and time-corrected peak areas, respectively, enabled accurate identification and quantification whether in standard solutions or in samples. Such performances were perfectly adapted to high-throughput routine determinations of organic acids in research or industry. Organic acids were assayed in different plant tissues and cells, including sycamore, arabidopsis, buttercup, and pea. Citrate and malate were the most abundant in all plants tested with concentrations reaching 18.9 and 22.3 micromol/g fresh matter, respectively. Cadmium effect on pea leaves metabolism was also assessed.

Carboxylic Acids↗
PubMed1998

Specific oligonucleotide primers for the direct detection of plum pox potyvirus-cherry subgroup.

A specific polymerase chain reaction assay was developed for direct identification of the distinct subgroup of plum pox potyvirus (PPV) isolates from cherry trees (PPV-cherry, PPV-C) and its differentiation from other known subgroups of PPV. The specificity of the assay is based on using a pair of primers whose nucleotide sequences are located on the coat protein gene of PPV-sour cherry (SoC) at regions of high nucleotide divergence between PPV-SoC and other isolates of PPV. The technique will be useful for studying the epidemiology of PPV-C as well as for practical testing in certification and quarantine programs worldwide.

DNA Primers↗
PubMed2005

A composite-likelihood approach for detecting directional selection from DNA sequence data.

We present a novel composite-likelihood-ratio test (CLRT) for detecting genes and genomic regions that are subject to recurrent natural selection (either positive or negative). The method uses the likelihood functions of Hartl et al. (1994) for inference in a Wright-Fisher genic selection model and corrects for nonindependence among sites by application of coalescent simulations with recombination. Here, we (1) characterize the distribution of the CLRT statistic (Lambda) as a function of the population recombination rate (R=4Ner); (2) explore the effects of bias in estimation of R on the size (type I error) of the CLRT; (3) explore the robustness of the model to population growth, bottlenecks, and migration; (4) explore the power of the CLRT under varying levels of mutation, selection, and recombination; (5) explore the discriminatory power of the test in distinguishing negative selection from population growth; and (6) evaluate the performance of maximum composite-likelihood estimation (MCLE) of the selection coefficient. We find that the test has excellent power to detect weak negative selection and moderate power to detect positive selection. Moreover, the test is quite robust to bias in the estimate of local recombination rate, but not to certain demographic scenarios such as population growth or a recent bottleneck. Last, we demonstrate that the MCLE of the selection parameter has little bias for weak negative selection and has downward bias for positively selected mutations.

Base Sequence↗
PubMed1995

PCR for direct detection of indigenous uncultured magnetic cocci in sediment and phylogenetic analysis of amplified 16S ribosomal DNA.

PCR primers specific to the 16S ribosomal DNA (rDNA) of magnetic cocci were designed and used to amplify DNA from magnetically isolated magnetic cocci. The PCR products were subcloned by ligation into plasmid vector pCRII, and five clones containing approximately 270-bp fragments of amplified DNA were sequenced. The specific primers were also used to detect magnetic coccus 16S rDNA in environmental samples. Magnetic coccus 16S rDNA was amplified from the water column above sediment kept in an anoxic environment in the laboratory, but little was amplified from a water column kept in an oxic environment. These results suggest that magnetic cocci in the water column in an anoxic environment had migrated there from the sediment as a response to the microoxic or anoxic conditions, rather than having been present previously in a nonmagnetic form and having become magnetic due to these conditions. The specific primers were also used to detect magnetic cocci in aquatic sediment. DNA was extracted from sediment by direct lysis and purified for use as a PCR template by electrophoresis on an agarose-polyvinylpyrrolidone gel. 16S rDNA was then amplified and subcloned, and two clones were sequenced. The clones were screened for chimeric DNA by comparing sections of each with the GenBank database.

Bacteria↗
PubMed1993

Direct detection of major histocompatibility complex class I binding to antigenic peptides using surface plasmon resonance. Peptide immobilization and characterization of binding specificity.

We have developed model systems in which the binding of purified, genetically engineered, soluble analogues of major histocompatibility complex (MHC) class I molecules to immobilized antigenic peptides can be monitored in real time using surface plasmon resonance (SPR). Synthetic analogues of several peptides known to bind different mouse and human MHC class I molecules were prepared with cysteine residues substituted at appropriate positions. The analogue peptides were immobilized via the bifunctional reagent N-gamma-maleimidobutyryloxy-succinimide to amino groups generated on the dextran-modified gold surface of a biosensor flow cell. Using this approach, each position in the sequence of an H-2Ld-specific viral peptide, pMCMV (YPHFMPTNL), was used for coupling, and the resulting surfaces were tested for binding of the soluble analogue of H-2Ld, H-2Lds. In accord with our previously described H-2Ld/pMCMV three-dimensional structural model, only those residues of the peptide that remain exposed following binding (positions 4-8) can be replaced by cysteine and used for coupling. Stable binding of soluble MHC class I molecules, H-2Lds, H-2Dds, H-2Kbs, and HLA-A2s to their respective immobilized cognate peptides was detected by SPR. Specificity of the peptide/MHC interaction was characterized both by direct binding using immobilized peptides and by competition with peptides in solution, and in general was consistent with known immunological reactivity. Some peptides bound not only their cognate MHC molecule, but others at lower apparent affinity. Measurement of real time binding of MHC class I molecules to peptides immobilized through specific side chains suggests the application of a similar approach to the study of the interaction of peptides with a wide variety of peptide-binding macromolecules.

Alleles↗
PubMed1986

[Direct detection of Chlamydia trachomatis with monoclonal antibodies].

A total of 571 specimens obtained from the Heidelberg outpatient clinic for sexually transmitted diseases (STD) and 254 specimens sent in by mail were examined for Chlamydia trachomatis, using a direct immunofluorescence technique with monoclonal antibodies and tissue culture. With respect to tissue culture, the sensitivity of specimens from male STD patients was 77% and specificity was 99%; for specimens from female STD patients the corresponding values were 77% and 96%, respectively. In female prostitutes the sensitivity of specimens reached only 55%, and specificity in this group was 96%. Follow-up of the patients with discrepant results confirmed the results of the test, which had initially indicated the chlamydial infection. In the material sent in by mail, the detection rate of C. trachomatis by direct immunofluorescence with monoclonal antibodies was significantly higher than in tissue culture. When specimens have to be transported long distances, the direct test with monoclonal antibodies has proved to be superior to tissue culture. However, the advantage of this test (Micro Trak) is counterbalanced by a relatively low sensitivity.

Antibodies, Monoclonal↗
PubMed1999

Direct detection of intratumoral 5-fluorouracil trapping using metabolic 19F MR imaging.

The effective use of 5-fluorouracil (5-FU) in cancer therapy requires the noninvasive assessment of its transport, metabolism, and retention ("trapping") in the different tissues of the organism, particularly in the tumor. We used a chemical-shift selective 19F magnetic resonance (MR) imaging technique to map selectively 5-FU and its major catabolite alpha-fluoro-beta-alanine (FBAL) in six ACI rats bearing Morris hepatoma. After i.v. administration of 200 mg/kg-bw 5-FU, three metabolic MR maps were acquired consecutively in each animal: 1) an early 5-FU image (5-37 min post-injection (p.i.); dominant Fourier line, 8 min p.i.) characterizing the early uptake of 5-FU into the various tissues; 2) an FBAL image (40-72 min p.i.; dominant Fourier line, 56 min p.i.) reflecting the catabolism of the drug; and 3) a late 5-FU image (75-107 min p.i.; dominant Fourier line, 78 min p.i.) to assess the retention of unmetabolized 5-FU and its MR-visible anabolites. In the early 5-FU maps, the drug was detected in all major organs (e.g., heart, liver, kidneys) as well as in the muscular system. The FBAL maps showed no FBAL accumulation in the hepatoma which reveals that the tumor cells have lost hepatocellular functions relevant for 5-FU catabolism. On the late 5-FU maps, a significant amount of 5-FU was detected in only one of the six Morris hepatomas. The observation in this rat verifies directly that 5-FU can be trapped in solid tumors. The images, moreover, emphasize the necessity of acquiring spatially-resolved MR data to detect metabolic tumor heterogeneity.

Animals↗
PubMed2001

Direct detection of mecA, nuc and 16S rRNA genes in BacT/Alert blood culture bottles.

A benzyl alcohol-guanidine hydrochloride extraction method was used to remove sodium polyanetholesulfonate present in BacT/Alert blood culture bottles. Multiplex PCR using touchdown annealing was used to detect the mecA, nuc, and 16S rRNA genes in bottles growing staphylococci. This direct PCR assay demonstrated excellent sensitivity, specificity and improved accuracy compared to routine phenotypic methods for determination of methicillin resistance in coagulase negative staphylococci (CoNS). However, with this PCR assay, bottles that contained both methicillin-resistant CoNS and methicillin-susceptible Staphylococcus aureus would be misidentified as containing methicillin-resistant S. aureus.

Bacterial Proteins↗
PubMed1997

Prenatal diagnosis of rubella virus infection by direct detection and semiquantitation of viral RNA in clinical samples by reverse transcription-PCR.

A reverse transcription-nested PCR (RT-nPCR) method for prenatal diagnosis of rubella virus (RV) infection was developed. In the first step of RT-nPCR a synthetic RNA molecule (pRRV) differing from the RV target sequence by having a 21-nucleotide insertion was used as the internal control of amplification for the detection of PCR inhibitors. In addition, comparison of pRRV and RV-specific PCR signals allowed for the semiquantitation of RV input target sequences (range, 10 to > and = 1,000 RV genomes). In parallel, a complete RT-nPCR assay was performed with the same samples in the absence of the internal control to confirm the results of the first step and to detect RV RNA-positive samples containing < 10 RV genomes. Subsequently, the RT-nPCR method was used to examine retrospectively clinical samples (direct RT-nPCR) from eight congenitally infected and eight uninfected fetuses for RV RNA. RT-nPCR was also used to detect RV RNA in cell cultures (culture-RT-nPCR) 96 h after inoculation with the same specimens. With amniotic fluid (AF) samples, direct RT-nPCR identified eight of eight cases of RV transmission (sensitivity, 100%), whereas culture-RT-nPCR and virus isolation detected only six of eight cases (sensitivity, 75%). However, when the culture-RT-nPCR results were positive, culture-RT-nPCR confirmed the direct RT-nPCR results 3 days to 3 weeks earlier than virus isolation. The specificity of direct RT-nPCR was 100%, with eight of eight uninfected fetuses being negative. Semiquantitation showed only small amounts (< and = 100 copies) of viral RNA in clinical samples. In conclusion, direct RT-nPCR with AF samples (i) shows 100% sensitivity and specificity for prenatal diagnosis of RV infection and (ii) is a rapid technique, giving results in 24 to 48 h after sampling.

Base Sequence↗
PubMed2005

Solid sampling technique for direct detection of condensed tannins in bark by matrix-assisted laser desorption/ionization mass spectrometry.

A novel method for the direct analysis of condensed tannin components in bark was developed on the basis of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) combined with a simple solid sampling technique. The MALDI mass spectra obtained from the wood (bark) powder sample clearly showed a series of peaks corresponding to the sodium ion adducts of condensed tannin oligomers up to around m/z 3000. The results indicate that the condensed tannins in the bark sample used in this work mostly consist of combinations of flavan-3-ol units such as profisetinidin (PF), prorobinetinidin (PR) and prodelphinidin (PD), at least up to 10-mers (m/z approximately 3000).

Acacia↗