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PubMed1996

Direct detection of disomy in human sperm by the PRINS technique.

We report the use of the PRimedIN Situ (PRINS) labelling technique for the direct estimation of disomy rates for various chromosomes in human sperm. The PRINS technique provides a rapid and reliable method for chromosome screening since specific labelling may be obtained in less than 2 h. In the present study, the disomy rates of chromosomes 2, 5, 9, 12 and 18 were investigated. The incidences of disomy are similar for all these chromosomes, ranging from 0.27% to 0.33%. These findings suggest an equal distribution of aneuploidies among autosomes in human sperm.

Aneuploidy↗
PubMedDate not supplied

Immunodetection by quartz crystal microbalance. A new approach for direct detection of rabbit IgG and peroxidase.

Biodetection is one of the most important challenges for the twenty-first century: many fields are concerned, mainly environmental and medical. The quartz crystal microbalance (QCM) may offer great possibilities for this purpose: a direct response signal, which characterizes the binding event between a sensitive layer, immobilized onto the surface transducer, and the analyte to be detected, can be obtained. However, for the detection of small biomolecules such as antigens, it is quite difficult to obtain an observable signal that corresponds directly to the binding event. In general, this is owing to the lack of mass sensitivity of the commonly used QCM, with 5- to 10-MHz quartz crystals. For improving this mass sensitivity, a 27-MHz quartz resonator was developed and incorporated in a flow-through microcell. Two biospecies, IgG rabbit and peroxidase enzyme, were studied with this ultra-sensitive QCM in terms of specificity, detection limit, and calibration curve.

Animals↗
PubMed1989

Use of a non-radioactive hybridisation assay for direct detection of gram-negative bacteria carrying TEM beta-lactamase genes in infected urine.

DNA in infected urines from 81 patients with urinary tract infection was hybridised directly with a non-radioactive DNA probe specific for bacterial genes coding for TEM-type beta-lactamase. The results were assessed by means of a computerised image analysis system and compared with those obtained following isolation of the infecting organism, conventional sensitivity testing and isoelectric focusing (IEF) procedures for the detection of TEM-type beta-lactamase. Of the 27 ampicillin-resistant gram-negative organisms isolated in pure culture from the urines, 14 were shown by both hybridisation and IEF to carry a gene for TEM beta-lactamase production. Only four discordant results were obtained: three "false positive" direct hybridisation results, one due to urine pigmentation, and one, possibly, to a TEM beta-lactamase gene which was not being expressed, and one "false negative" result due to insufficient cell numbers in the urine. The system is capable of screening large numbers of samples and is applicable to any gene for which a suitable DNA probe is available.

Ampicillin Resistance↗
PubMed2004

Organogermanium reactive intermediates. The direct detection and characterization of transient germylenes and digermenes in solution.

Diphenylgermylene (Ph2Ge) and its Ge=Ge doubly bonded dimer, tetraphenyldigermene (6a), have been characterized directly in solution for the first time by laser flash photolysis methods. The germylene is formed via (formal) cheletropic photocycloreversion of 3,4-dimethyl-1,1-diphenylgermacyclopent-3-ene (4a), which is shown to proceed in high chemical (>95%) and quantum yield (phi = 0.62) by steady-state trapping experiments with methanol, acetic acid, isoprene, and triethylsilane. Flash photolysis of 4a in dry deoxygenated hexane at 23 degrees C leads to the prompt formation of a transient assigned to Ph2Ge (lambda(max) = 500 nm; epsilon(max) = 1650 M(-1) cm(-1)), which decays with second-order kinetics (tau approximately 3 micros), with the concomitant growth of a second transient species that is assigned to digermene 6a (tau approximately 40 micros; lambda(max) = 440 nm). Analogous results are obtained from 1,1-dimesityl- and 1,1-dimethyl-3,4-dimethylgermacyclopent-3-ene (4b and 4c, respectively), which afford Mes2Ge (tau approximately 20 micros; lambda(max) = 560 nm) and Me2Ge (tau approximately 2 micros; lambda(max) = 480 nm), respectively, as well as the corresponding digermenes, tetramesityl- (6b; lambda(max) = 410 nm) and tetramethyldigermene (6c; lambda(max) = 370 nm). The results for the mesityl compound are compared to the analogous ones from laser flash photolysis of the known Mes2Ge/6b precursor, hexamesitylcyclotrigermane. The spectra of the three germylenes and two of the digermenes are in excellent agreement with calculated spectra, derived from time-dependent DFT calculations. Absolute rate constants for dimerization of Ph2Ge and Mes2Ge and for their reaction with n-butylamine and acetic acid in hexane at 23 degrees C are also reported.

Journal Article↗
PubMed2006

Multiplex PCR-ELISA for direct detection of MRSA in nasal swabs advantageous for rapid identification of non-MRSA carriers.

In the study presented here 251 nasal swabs obtained from medical staff were directly investigated for MRSA using a commercial multiplex PCR system in parallel with conventional culture methods to determine the usefulness of PCR for rapid screening. Both methods identified 3.2% (8/251) of specimens as MRSA-positive; one sample was culture-positive only, and three were PCR-positive only. PCR correctly identified 215 of 239 (90%) negative swab samples, but one sample with weak cultural growth was not detected and was therefore considered false negative. The comparative sensitivity of culture versus PCR was 75% (9/12) versus 91.6% (11/12). Although PCR had a low positive predictive value (31.4%) its negative predictive value was high (99.5%). The results of this study indicate the multiplex PCR is suitable for the rapid identification of MRSA-negative individuals directly from nasal swabs in populations with a low MRSA prevalence, but positive results need to be confirmed by culture.

Enzyme-Linked Immunosorbent Assay↗
PubMed1989

Direct detection of human cytomegalovirus in urine specimens from renal transplant patients following polymerase chain reaction amplification.

A polymerase chain reaction (PCR) assay was used to amplify human cytomegalovirus (HCMV) directly from urine specimens taken from renal transplant patients. In serial urine samples from patients who had at least one specimen positive for HCMV; the PCR assay consistently detected the presence of HCMV DNA sequences, whereas virus detection by other tests such as enzyme-linked immunosorbent assay (ELISA), nonradioactive DNA hybridization assay, and virus isolation were variable. Of 37 specimens positive by PCR, 36 were positive by either ELISA, hybridization assay, or virus isolation. Infectious virus was detected in 13 of the 37 PCR-positive urines. HCMV DNA was detected by PCR in all samples that were positive for HCMV by either hybridization assay or virus isolation. The viral genome copy number was determined by PCR assay for several urine samples that were positive by virus isolation but negative for HCMV by ELISA or hybridization assay. Viral genome copy number estimates indicated the presence of HCMV at very low levels in these urines verifying the fidelity of the virus isolation procedures. The consistency of the PCR assay makes it an ideal method for detection of infection and monitoring antiviral drug therapy in patients infected with HCMV.

Antigens, Viral↗
PubMed2005

Direct detection of brain acetylcholine synthesis by magnetic resonance spectroscopy.

The cholinergic system is an important modulatory neurotransmitter system in the brain. Changes in acetylcholine concentration have been previously determined directly in animal models and human brain biopsy specimens, and indirectly, by the effects of drugs, in living humans. Here, we developed a method for direct determination of acetylcholine synthesis in living brain tissue. The method is based on administration of choline, enriched with carbon-13 (stable isotope) in the two methylene positions, and detection of labeled acetylcholine and all other metabolic fates of choline, by carbon-13 magnetic resonance spectroscopy. We tested this method in rat brain slices and found it to be specific for acetylcholine synthesis in both the cortex and hippocampus. This method is potentially useful as a research tool for exploring the cholinergic system role in cognitive processes and memory storage as well as in diseases in which the malfunction of the cholinergic system has been implicated.

Acetylcholine↗
PubMed1999

Direct detection of vanA and vanB genes in clinical specimens for rapid identification of vancomycin resistant enterococci (VRE) using multiplex PCR.

Surveillance for vancomycin resistant enterococci (VRE) by culture can be labour intensive and time consuming. We have developed a multiplex polymerase chain reaction (MPCR) which can be performed directly on the clinical specimen. The assay allows sensitive detection of enterococci with vanA - and vanB -mediated resistance to vancomycin. DNA was purified from stool and rectal specimens using the XTRAX(TM)DNA Extraction Kit (Gull Labs). Multiplex PCR amplified vanA and vanB targets were detected using a microtiter plate EIA. Two-hundred specimens were tested by routine culture and MPCR. Culture identified 44 VRE isolates and MPCR detected 38 of the 44 culture positives. Multiplex PCR detected three additional positive VRE specimens missed by culture for a sensitivity and specificity of 86.4 and 98.1%, respectively. When the presence of PCR inhibitors was addressed in the six culture positive/MPCR negative specimens, four additional VRE positive specimens were detected. Performing MPCR on the original specimens and on a 1:10 dilution of all specimens to minimize the effect of inhibitors gave a sensitivity and specificity of 95.5 and 98.1%, respectively. Multiplex PCR with confirmation by microtiter plate hybridization could be completed in 8 h compared with 24-48 h required for culture.

Bacterial Proteins↗
PubMed1988

Direct detection of groups A, C and G streptococci in clinical specimens by a trivalent colour test.

A simple trivalent colour test, developed for the rapid detection and identification of streptococci belonging to Lancefield groups A, C and G, was evaluated for sensitivity and specificity with cultures and when directly used with wound and throat swabs. In tests performed on cultures, all of 94 group A, 78 group C and 94 group G cultures were correctly identified. In direct tests on wound swabs, 49 of 52 group A, 17 of 19 group C and 48 of 51 group G streptococci were detected and correctly identified; no false positives were observed. With throat swabs from pharyngitis patients 34 of 36 group A, 3 of 6 group C and 5 of 8 group G streptococci gave positive results. Almost 10% of these swabs gave false positive reactions with the group C component of the test system. Samples taken from uninfected individuals indicated that the false positives were probably associated with blood group A. The test system gives rapid and reliable results with streptococcal cultures, but when directly applied to clinical samples the results must be interpreted with caution, particularly if the patient's blood group is not known.

Antigens, Bacterial↗
PubMed1982

Direct detection of the sulfur trioxide radical anion during the horseradish peroxidase-hydrogen peroxide oxidation of sulfite (aqueous sulfur dioxide).

The ESR spectrum of SO3- is observed directly during the oxidation of (bi)sulfite to sulfate by horseradish peroxidase. This radical exhibits a single line at g = 2.0031. The SO3-radical can be trapped with nitrosobenzene, yielding an ESR spectrum with coupling constants AN = 12.3 G,AHp = AHo = 2.4 G, and AHm = 0.9 G, and a g-value of 2.0053. SO3- is an intermediate in the two-step reduction of peroxidase Compound I by (bi)sulfite at physiological pH. At low pH, no SO3- is observed, which indicates a direct, one-step, two-electron reduction of Compound I. The pH at which the mechanism changes depends on the isoenzymes present. The radical reacts rapidly with oxygen as evidenced by the absence of an ESR spectrum when oxygen is present and by oxygen uptake measurements.

Electron Spin Resonance Spectroscopy↗
PubMed1996

Direct detection of bovine herpes virus-1 DNA from cell culture fluids using polymerase chain reaction.

A pair of oligomers of 20 and 23 bp were designed for amplifying a 381 bp sequence from glycoprotein IV gene of bovine herpesvirus 1. The primer pairs were used for amplifying genomic DNA of BHV-1 directly from cell culture fluids under different experimental conditions such as, untreated cell culture fluid, thermal denaturation and proteinase K treatment in presence of detergent. The results reveal that direct thermal denaturation of cell culture fluid is sufficient to detect the virus by polymerase chain reaction.

Animals↗
PubMed1992

Solid-phase polymerase chain reaction: applications for direct detection of enteric pathogens in waters.

The techniques in current use for detection of pathogens in environmental samples are restricted to those organisms whose replication in either culture media or cell culture is feasible. These methods lack the selectivity and sensitivity necessary for their unequivocal detection and identification. We have developed an assay for the detection of bacterial cells in large volumes of water. Low concentrations of cells containing target sequences were concentrated on membrane filters and were subjected to amplification directly using a stepwise polymerase chain reaction. This procedure, together with nucleic acid probes, has enhanced the limit of detection to the level of a single bacterial cell. This technique could be used for the detection of any bacteria or virus in water or air.

Base Sequence↗
PubMed1997

Comparative evaluation of two commercial amplification assays for direct detection of Mycobacterium tuberculosis complex in respiratory specimens.

Two commercial assays detecting the presence of Mycobacterium tuberculosis complex in clinical specimens by rRNA target amplification (Gen-Probe Amplified M. tuberculosis Direct Test [AMTD]) and PCR (Amplicor) were evaluated. The tests were applied to 327 digested, decontaminated respiratory specimens collected from 236 patients. Results were compared with those of acid-fast staining and culture. The combination of culture and clinical diagnosis was considered the "gold standard." A total of 60 specimens were collected from 27 patients with a diagnosis of pulmonary tuberculosis. Thirteen of these specimens were from patients receiving standard antituberculosis therapy and therefore were not included in the comparison. Of the remaining 47 specimens, 33 were smear positive, 40 were culture positive, 45 were AMTD positive, and 39 were Amplicor positive. After resolution of discrepant results, the overall sensitivities, specificities, and positive and negative predictive values were 77, 100, 100, and 95 for staining; 87, 100, 100, and 97.4 for culture; 95.9, 98.9, 94, and 99.2 for AMTD; and 85.4, 99.6, 97.9, and 97.1 for Amplicor, respectively. Agreement between AMTD and Amplicor assay results was 96.8%. It is concluded that although both nucleic acid amplification methods are rapid and specific for the detection of M. tuberculosis complex in respiratory specimens, AMTD appeared to be more sensitive than Amplicor.

Bacterial Typing Techniques↗
PubMed1991

Direct detection of yeast mutants with reduced viability on plates by erythrosine B staining.

In this paper we report a rapid method to screen yeast mutants exhibiting reduced viability directly on plates. This method avoids the need for replica plating and is based on the addition of the vital dye erythrosine B in nutrient medium. After 2 or 3 days of culture, colonies containing a large proportion of dead cells show a pink or a dark pink color whereas normal colonies are practically white.

Colony Count, Microbial↗
PubMed2003

Six rapid tests for direct detection of Clostridium difficile and its toxins in fecal samples compared with the fibroblast cytotoxicity assay.

Clostridium difficile is one of the most frequent causes of nosocomial gastrointestinal disease. Risk factors include prior antibiotic therapy, bowel surgery, and the immunocompromised state. Direct fecal analysis for C. difficile toxin B by tissue culture cytotoxin B assay (CBA), while only 60 to 85% sensitive overall, is a common laboratory method. We have used 1,003 consecutive, nonduplicate fecal samples to compare six commercially available immunoassays (IA) for C. difficile detection with CBA: Prima System Clostridium difficile Tox A and VIDAS Clostridium difficile Tox A II, which detect C. difficile toxin A; Premier Cytoclone A/B and Techlab Clostridium difficile Tox A/B, which detect toxins A and B; and ImmunoCard Clostridium difficile and Triage Micro C. difficile panels, which detect toxin A and a species-specific antigen. For all tests, Triage antigen was most sensitive (89.1%; negative predictive value [NPV] = 98.7%) while ImmunoCard was most specific (99.7%; positive predictive value [PPV] = 95.0%). For toxin tests only, Prima System had the highest sensitivity (82.2%; NPV = 98.0%) while ImmunoCard had the highest specificity (99.7%; PPV = 95.0%). Hematopoietic stem cell transplant (HSCT) patients contributed 44.7% of all samples tested, and no significant differences in sensitivity or specificity were noted between HSCT and non-HSCT patients. IAs, while not as sensitive as direct fecal CBA, produce reasonable predictive values, especially when both antigen and toxin are detected. They also offer significant advantages over CBA in terms of turnaround time and ease of use.

Bacterial Toxins↗
PubMed2000

Development and modification of a recombinant cell bioassay to directly detect halogenated and polycyclic aromatic hydrocarbons in serum.

Polycyclic and halogenated aromatic hydrocarbons (PAHs/HAHs) are a diverse group of widespread and persistent environmental contaminants that can cause a variety of detrimental effects in vertebrates. As most available methods to detect these contaminants are expensive, labor and time intensive, and require large amounts of tissue for extraction and analysis, several rapid mechanistically based bioassay systems have been developed to detect these chemicals. Here we describe application and optimization of a recently developed recombinant mouse cell bioassay system that responds to both PAHs and HAHs with the rapid induction of firefly luciferase for the detection of these chemicals in whole serum samples. This chemically activated luciferase expression (CALUX) bioassay has been modified to allow rapid (4-h) and direct analysis of small volumes (25-50 microl) of whole serum in a 96-well microtiter plate format without the need for solvent extraction. This bioassay can detect as little as 10 parts per trillion of the most potent HAH, 2,3,7,8-TCDD, and is also sensitive to other HAHs and PAHs. The use of simple procedures corrects for interplate and intraplate variability and the Ah receptor dependence of the induction response is accounted for by use of the antagonist 4-amino-3-methoxyflavone.

Algorithms↗
PubMed2000

Comparison of enhanced Mycobacterium tuberculosis amplified direct test with COBAS AMPLICOR Mycobacterium tuberculosis assay for direct detection of Mycobacterium tuberculosis complex in respiratory and extrapulmonary specimens.

The new Roche COBAS AMPLICOR Mycobacterium tuberculosis Assay was compared to the Gen-Probe enhanced Mycobacterium tuberculosis Amplified Direct Test (AMTDII). A total of 486 specimens (296 respiratory and 190 extrapulmonary) collected from 323 patients were tested in parallel with both assays. Results were compared with those of acid-fast staining and culture, setting the combination of culture and clinical diagnosis as the "gold standard." After resolution of discrepant results, the sensitivity, specificity, and positive and negative predictive values for AMTDII were 85.7, 100, 100, and 90.4% for respiratory specimens and 82.9, 100, 100, and 95. 5% for extrapulmonary specimens, respectively. The corresponding values for AMPLICOR were 94.2, 100, 100, and 96.6% for respiratory specimens and 85, 100, 100, and 96.1% for extrapulmonary specimens, respectively. No significant differences were observed between the results of both assays or, within each one, between respiratory and extrapulmonary specimens. The difference between AMTDII and AMPLICOR sensitivities was related to the presence of inhibitory samples, which the former assay, lacking an internal amplification control (IAC), could not detect. The overall inhibition rate for the AMPLICOR assay was 3.9% (19 specimens). It is concluded that, although both amplification assays proved to be rapid and specific for the detection of M. tuberculosis complex in clinical samples, AMPLICOR, by a completely automated amplification and detection procedure, was shown to be particularly feasible for a routine laboratory setting. Finally, AMTDII is potentially an excellent diagnostic technique for both respiratory and extrapulmonary specimens, provided that an IAC is included with the assay.

Biopsy↗