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PubMed1983

Rapid and direct detection of herpes simplex virus and varicella-zoster virus antigens in clinical specimens by staphylococcal reagent and membrane filtration.

Herpes simplex virus was detected rapidly and directly in 31 and varicella-zoster virus in two of 50 clinical specimens using specific antisera, stabilized staphylococci rich in protein A and membrane filtration. Microscopical examination of the cells retained on the filter membrane revealed attached staphylococci only on cells harbouring viral antigens but not on non-infected cells or cells from healthy donors. Infected cells treated with negative control sera and stabilized staphylococci and subsequently subjected to membrane filtration were also devoid of the marker. It was also possible to detect herpes simplex virus antigens in three specimens which were culture negative. Similar results were obtained with staphylococci specifically sensitized with anti-herpes simplex rabbit serum. No interfering, non-specific background of unattached staphylococci was observed on the filter membrane. The results were confirmed by the direct immunofluorescent test. The method is sensitive, specific, and provides results within 3 h. It could be employed for the rapid screening of populations at risk, e.g. pregnant women, medical personnel, and societies with a growing incidence of genital herpes. Since no special, expensive equipment is required, the method is also suitable for modestly equipped clinical laboratories.

Antibodies, Viral↗
PubMed2001

Polymerase chain reaction for the direct detection of Brucella spp. in milk and cheese.

A polymerase chain reaction test was developed to detect Brucella spp. directly in milk and cheese and optimized using primers for the BSCP-31 gene. A total of 46 cheese samples produced with sheep and goats milk were assayed, and Brucella spp. was detected in 46% of them, especially in cheese made from sheep milk. This method is of remarkable epidemiologic interest because it is an indirect test indicating the sanitary quality of milk used in dairy industries. The method showed good sensitivity and specificity. It is faster and less expensive than the conventional bacteriological assays.

Animals↗
PubMed2003

Direct detection of deletion mutations in the yeast DEL assay using quantitative PCR (TaqMan).

Established mutagenesis assays measure mutant frequencies at selectable loci. These assays work by encouraging the growth of mutants to form visible colonies while suppressing the growth of non-mutants. An alternative strategy is to detect DNA alterations directly. We present an example of this latter strategy using TaqMan to quantify deletion mutants in mixed cultures of Saccharomyces cerevisiae strain RS112. The RS112 strain contains two heteroallelic his3 sequences that share approximately 400bp homology and are separated by approximately 7 kb of plasmid DNA. Spontaneous and chemical-induced strand breaks that occur in this region are repaired by intrachromosomal recombination, resulting in the loss of the plasmid DNA and creation of a His prototroph. Ordinarily, these prototrophs are detected by growth on His- medium over 2-3 days. In this case, we used TaqMan to selectively detect the DNA of deletion mutants in the presence of a large excess of DNA from non-mutants. This was accomplished using primers whose annealing sites were outside the region of DNA lost due to recombination. Thus, the primers were too far apart to produce PCR products using DNA from non-mutants, but produced a robust TaqMan signal using DNA from deletion mutants. Spontaneous and chemical-induced recombination frequencies (RF) were measured in a series of time-course and dose-response experiments with direct-acting mutagens. Interestingly, chemical-induced increases in RF were observed within a few hours of initiation of exposure, demonstrating that deletion mutations in RS112 can be fixed soon after DNA damage occurs. The ability to measure RF at any time during treatment will be useful for additional mechanistic studies. Chemical-induced increases in RF were also observed in the absence of selective growth conditions. As such, detection of deletion mutations with TaqMan may be applicable to measurements of RFs at non-selectable loci in yeast and other species. Finally, chemical-induced RFs after 17 h exposure were similar to those observed after 3 days growth on selective medium. The TaqMan assay may therefore be used to screen compounds more quickly for their ability to cause deletion mutations than is currently done by plating on selective medium.

Base Sequence↗
PubMed2005

A sensitive immunoassay based on electropolymerized films by capacitance measurements for direct detection of immunospecies.

Fabrication of a capacitive immunosensor based on electropolymerized polytyramine (Pty) film for the direct detection of human serum albumin (HSA) without any labeling is described. The capacitance change of the heterostructures, Pty films/covalently bonded antibodies/buffered medium, is utilized for monitoring the specific antibody-antigen interaction. The Pty films are ultrathin and the HSA assay is nearly specific. Experimental parameters affecting antibody immobilization and the sensing of HSA are investigated in detail and optimized. This capacitive sensor prepared with the present method can provide high sensitivity. Under the optimized experimental conditions, a linear calibration curve in the concentration range 1.84-368.6 ng/ml when plotted vs the logarithm of the antigen concentration is obtained and the detection limit (S/N=3) is 1.60 ng/ml. After an acidic washing the present system can be used again. The applicability and reliability of the sensor are also demonstrated.

Animals↗
PubMed1999

PCR identification of Pseudomonas aeruginosa and direct detection in clinical samples from cystic fibrosis patients.

This report describes a PCR primer pair that targets the algD GDP mannose gene of Pseudomonas aeruginosa and produces a specific 520-bp PCR product useful for P. aeruginosa identification. This PCR assay was tested with 182 isolates of P. aeruginosa and 20 isolates of other bacterial species, and demonstrated 100% specificity and sensitivity. The test was also able to detect P. aeruginosa directly in clinical samples such as sputum or throat swabs obtained from cystic fibrosis patients. The combination of this primer with a universal bacterial primer, acting as a control to assess DNA quality in the sample, resulted in a robust PCR method that can be used for rapid P. aeruginosa detection.

Carbohydrate Dehydrogenases↗
PubMed2004

[Direct detection of rifampicin resistant Mycobacterium tuberculosis in sputum by line probe assay (LiPA)].

OBJECTIVES: To examine the direct detection of rifampicin (RFP)-resistant Mycobacterium tuberculosis in sputum by Line Probe Assay (LiPA). MATERIALS AND METHODS: We collected 130 sputa and analyzed both by LiPA and the Amplicor M.tuberculosis assay. For culture-positive samples, RFP resistance testing was performed and compared with the results by LiPA. RESULTS: Eighty two out of 84 M. tuberculosis samples were detected by LiPA and all of 10 Mycobacteria other than M. tuberculosis (MOTT) samples and 36 negative samples were negative by LiPA. The detection rate is same as Amplicor. For culture-positive samples, LiPA showed mutation pattern for all of 22 RFP-resistant strains and wild type pattern for 19 of 20 RFP-sensitive strains. The one remaining showed mixed pattern of wild type and mutation pattern. CONCLUSION: The use of LiPA for sputum coould enable early detection of RFP-resistant tuberculosis and seems to be useful for the control of tuberculosis.

Antibiotics, Antitubercular↗
PubMed1998

Evaluation of ligase chain reaction for direct detection of Mycobacterium tuberculosis in respiratory specimens.

A new automated amplification method, Ligase Chain Reaction (LCx MTB), was evaluated for direct detection of Mycobacterium tuberculosis in respiratory specimens from 208 patients and its performance was compared with culture and direct smears. Out of 226 specimens, 28 LCx MTB and 15 cultures were found positive for M. tuberculosis. After resolution of clinical history, the sensitivity of LCx MTB and culture was respectively 89.3% and 53.6% with a specificity of 98.5% and 100%. However, samples coming from untreated patients presented similar results between culture and LCx MTB (sensitivity 75% and 83.3% for culture and LCx MTB).

Automation↗
PubMedDate not supplied

A metal-chelating piezoelectric sensor chip for direct detection and oriented immobilization of polyHis-tagged proteins.

A metal-chelating piezoelectric (PZ) chip for direct detection and controlled immobilization of polyHis-tagged proteins has been demonstrated. The chip was prepared by covalently binding a hydrogel matrix complex of oxidized dextran and nitrilotriacetic acid (NTA) ligand onto an activated alkanethiol-modified PZ crystal. The resulting chip effectively captured Ni2+ ions onto its NTA surface, as disclosed by the resonant frequency shift of the crystal and an X-ray photoelectron spectroscopy analysis. The real-time frequency analysis revealed that the bare NTA chip was nonfouling, regenerable, and highly reusable during continuous repetitive injections of ion solutions and binding proteins. In addition, the chip displayed good long-term reusability and storage stability. The individual binding studies of a polyHis-tagged glutathione-S-transferase and its native untagged form on various metal-charged chips revealed that Co2+, Cu2+, and Ni2+ ions each had different immobilization ability on the NTA surface, as well as their binding ability and selectivity with the tagged protein. As a result, the tagged protein immobilized on the Ni2+-charged chip can actively be bound with its antibody and substrate. Further, the quantitative analyses of the tagged protein in crude cell lysate with a single Ni2+-charged chip and of its substrate with a protein-coated chip were also successfully demonstrated. Therefore, this study initiates the possibilities of oriented, reversible, and universal immobilization of any polyHis-tagged protein and its functional study using a real-time PZ biosensor.

Biosensing Techniques↗
PubMed1994

Development and evaluation of rapid monoclonal antibody-based coagglutination test for direct detection of Vibrio cholerae O139 synonym Bengal in stool samples.

A monoclonal antibody-based coagglutination test directly detected Vibrio cholerae O139 synonym Bengal in 83 of 120 watery diarrheal stool specimens; on culture, 90 samples were positive. Thus, with 92% sensitivity, 100% specificity, and 100% positive and 95% negative predictive values, the coagglutination test is a useful rapid test for V. cholerae O139.

Agglutination Tests↗
PubMed2006

Novel applications of highly sensitive liquid chromatography/mass spectrometry/mass spectrometry for the direct detection of ultra-trace levels of contaminants in water.

Recent advances in the sensitivity of liquid chromatography/mass spectrometry (LC/MS) instrument technology provide the basis for the direct detection, i.e. without sample pre-concentration, of organic contaminants in water in the ng/L range. Novel applications for the analysis of atrazine and some of its desalkylated and hydroxylated degradation products, the pharmaceutical compounds diclofenac and carbamazepine, sulfonylurea herbicides, and iodinated X-ray contrast media have been developed. For each analyte a specific tandem mass spectrometric (MS/MS) transition has been selected and the corresponding mass spectrometric parameters optimised. All analytes could be analysed within three specific analytical runs including different high-performance liquid chromatography (HPLC) conditions. Detection limits were determined to be better than 10 ng/L for the direct analysis of the compounds in water except for X-ray contrast media, for which detection limits were found to be up to one order of magnitude higher. The methods have been successfully utilised for the analysis of natural waters. Matrix effects frequently occurring in LC/MS have shown to be low to moderate in the case of X-ray contrast media. This work demonstrates that for the analysis of a large number of water contaminants, the sample pre-concentration step could possibly be omitted.

Chromatography, High Pressure Liquid↗
PubMedDate not supplied

Evaluation of the upgraded amplified Mycobacterium tuberculosis direct test (gen-probe) for direct detection of Mycobacterium tuberculosis in respiratory and non-respiratory specimens.

We evaluated the upgraded Amplified Mycobacterium Tuberculosis Direct Test kit (AMTD) (Gen-Probe Inc.) for the direct detection of Mycobacterium tuberculosis in respiratory and non-respiratory specimens, and compared the results between the traditional 30,000 RLUs cutoff criteria (C) and three equivocal ranges (30,000-100,000, R1; 30,000-500,000, R2; and 30,000-1,000,000, R3). We tested 663 respiratory and 238 non-respiratory samples from 464 patients. The gold standard was considered to be the combination of culture and clinical data. One hundred and nineteen samples were from 56 patients with pulmonary tuberculosis, and 36 samples were from 19 patients with extrapulmonary tuberculosis. When C criteria was applied, the sensitivity and specificity values were 90.8 and 93.0% for respiratory specimens, while they were 88.9 and 92.1% for non-respiratory specimens (p = NS). The sensitivity was significantly higher in smear-positive specimens (96.7%) than in smear-negative ones (81.0%) (p < 0.05). When compared with C criteria, the overall sensitivity was maintained at 90.3% for R1 criteria, and slightly decreased to 89.7% for R2 and R3 criteria (p = NS). Overall, specificity increased significantly from 92.9% (C) to 97.5% (R1), 99.1% (R2), and 99.2% (R3). Application of R2 or R3 criteria improved significantly the specificity of the test with little decrease in sensitivity.

Humans↗
PubMedDate not supplied

Determination of absolute configurations of light-atom molecules by means of direct detection of Bijvoet differences.

The absolute configurations of two light-atom molecules were determined sufficiently well by direct detection of Bijvoet differences. The compounds examined were (I) beta-cytidine C9H13N3O5 and (II) (S)-3-[(R)-4,4,4-Trifluoro-3-[4-methoxyphenyl]butanoyl]-4-(phenylmethyl)oxazolidin-2-one, C21H20NO4F3. Both compounds crystallize in orthorhombic system with the space group P2(1)2(1)2(1) and Z = 4. The crystal structures were carefully refined by the technique of conventional structure analysis. All possible reflections were measured on a laboratory diffractometer with Cu Kalpha radiation. The multiple-diffraction effect was often observed especially as remarkable intensity enhancement in weak reflections. After such unreliable reflections were eliminated by comparisons among the equivalent mates, data were averaged to a set of Bijvoet pairs. Afterwards, additional measurements by the psi-scan technique were tested. Since psi-scan data showed a slight systematic error probably owing to some shape-effect, an artificial absorption correction DIFABS was adopted to remove the error. Small but significant intensity differences could be detected for many Bijvoet pairs, and the absolute configurations were correctly determined without ambiguity in all cases. The R and wR values for separate refinements of enantiomorphs supported the results with slight differences. Flack parameters indicated no contradictions as well.

Journal Article↗
PubMedDate not supplied

Direct detection of drugs of abuse in whole hemolyzed blood using the EMIT d.a.u. urine assays.

A simple, rapid, and sensitive method for the direct detection of a broad spectrum of drugs of abuse in hemolyzed whole blood by the enzyme multiplied immunoassay technique (EMIT) is described. A methanolic extract of 1 mL of whole blood is directly analyzed with EMIT urine assays. The proposed method is very sensitive and can detect drug concentrations in low therapeutic to subtherapeutic concentration ranges for all ten assays used. The EMIT urine assays used in this study included those for opiates, amphetamines, methadone, barbiturates, phencyclidine (PCP), methaqualone, propoxyphene, cocaine metabolite, benzodiazepine metabolite, and cannabinoids. This method should be most useful for screening forensic cases by EMIT when no urine is available, such as in impaired driving cases.

Amphetamines↗
PubMed1994

Direct detection of Listeria monocytogenes using paramagnetic bead DNA extraction and enzymatic DNA amplification.

The potential of the polymerase chain reaction (PCR) as a tool for direct detection of Listeria monocytogenes in food and clinical samples was investigated. A specific oligonucleotide, directed against the listeriolysin 0 gene of L. monocytogenes, was coupled to paramagnetic beads and used to isolate listerial DNA from food homogenates and blood. The isolated DNA was amplified with a seminested PCR procedure, which recognized the hly gene. The detection limit for L. monocytogenes in 50 ml of a buffer solution was between 1 and 10 colony forming units (cfu). In food homogenates consisting of 10 g food and 40 ml 0.85% NaCl solution, artificially spiked with an overnight culture of L. monocytogenes, the detection limit was 1-10 cfu. The method was further evaluated by application to naturally contaminated food samples. In 250 microliters whole human blood artificially spiked with L. monocytogenes 10,000 cfu could be detected.

Base Sequence↗
PubMed2004

"Direct" detection and separation of DNA using nanoporous alumina filters.

The concept of using alumina nanoporous filters (AAO) modified with DNA for "label-free" detection and separation/purification of the target ss-DNA is demonstrated. The high surface density of DNA (4 x 10(12) cm(-2)) and high efficiency of hybridization (ca. 70%) in combination with increased effective surface area make this system very attractive for development of various ss-DNA (or RNA) detection methods. Moderate transparency of AAO in the UV and IR regions allows direct detection of DNA hybridization by optical and IR absorption. Close to the quantitative efficiency of binding the target ss-DNA from solution using a single pass through the modified filter is observed.

Aluminum Oxide↗
PubMed1999

Evaluation of the Abbott LCx Mycobacterium tuberculosis assay for direct detection of Mycobacterium tuberculosis complex in human samples.

Seven hundred thirty-seven clinical samples from 460 patients were processed for direct detection of Mycobacterium tuberculosis complex by a semiautomated ligase chain reaction commercial assay, the LCx Mycobacterium tuberculosis Assay (LCx assay) from Abbott Laboratories. Results were compared to those of direct microscopy and standard microbiological culture. Of 26 patients (5.7%) with a culture positive for M. tuberculosis, 22 (84.6%) were found positive by the LCx assay. The sensitivity of the LCx assay was 98% for smear-positive samples and 27% for smear-negative samples. With an overall culture positivity rate for M. tuberculosis of 8.3% (61 of 737 samples) and after resolution of discrepant results according to clinical data, the sensitivity, specificity, and positive and negative predictive values of the LCx assay were 78, 100, 95, and 98%, respectively, compared to 85, 100, 100, and 98%, respectively, for culture and 67, 99, 87, and 97%, respectively, for acid-fast staining. In conclusion, the LCx assay proved satisfactory and appears to be an easy-to-use 1-day test which must be used with standard culture methods but can considerably reduce diagnosis time versus culture. However, its clinical interest appears to be limited in our population with low mycobacterial prevalence because of its cost considering the small gain in sensitivity versus direct microscopy.

Bacteriological Techniques↗
PubMed1999

Direct detection of bacteria in cellular blood products using bacterial ribosomal RNA-directed probes coupled to electrochemiluminescence.

Various techniques for detecting bacteria in blood products exist; however, none has been widely accepted. Our method permits direct bacterial detection in both platelet concentrates (PC) and packed red blood cells (RBC). This novel procedure targets bacterial ribosomal RNA (rRNA) but does not utilize culture or nucleic acid amplification. The assay comprises five steps: (1) release of bacterial rRNA by cell lysis with a combination of detergents and high heat; (2) hybridization of bacterial rRNA using a biotin- and a ruthenium (ORIGEN)-labelled oligonucleotide probe pair; (3) capture of labelled rRNA with streptavidin-coated magnetic beads; (4) concentration of labelled rRNA/bead complexes out of solution and onto an electrode surface with a magnet; (5) detection of ruthenium-labelled bacterial rRNA by application of voltage and consequent generation of the electrochemiluminescent (ECL) signal. Results using PC and RBC samples, spiked with clinically relevant gram-negative and -positive bacterial species, consistently demonstrated a linear relationship between ECL signal (equates to rRNA level) and colony forming units (CFU) mL(-1). Signals were generated in the range of 1400-80000 and 3500-500000 ECL units for unwashed and washed samples, respectively. This is equivalent to 10(5)-10(8)CFU mL(-1). These data demonstrate that therapeutic blood products significantly contaminated with bacteria may be identified prior to issue.

Bacteria↗