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PubMed1995

A new plasmid cloning vector for direct detection of recombinant clones, based on inactivation of a bacterial acid phosphatase-encoding gene.

A new plasmid cloning vector for Escherichia coli (pPhoR) suitable for direct detection of recombinant clones has been constructed. The plasmid is a multicopy of a vector which carries a pUC-derived origin of replication and beta-lactamase gene, and the phoN acid phosphatase-encoding gene from Providencia stuartii. Foreign DNA fragments can be cloned into unique restriction sites located within the phoN gene causing a loss of the acid phosphatase activity which is normally overproduced by E. coli strains carrying pPhoR. Since PhoN production can be easily detected by a plate histochemical assay, recombinant clones carrying foreign DNA fragments inserted in the phoN gene can be easily detected as PhoN-negative clolonies on the above medium. The efficiency of the pPhoR-based cloning system for direct cloning of PCR amplimers of a variable region of the HIV-1 genome was comparable to that of conventional cloning systems for direct detection of recombinant based on beta-galactosidase inactivation. Advantage of the pPho-R-based system include reduced costs for histochemical assays and the possibility of being used with any E. coli host.

Acid Phosphatase↗
PubMed1996

The development of a new biosensor based on recombinant E. coli for the direct detection of organophosphorus neurotoxins.

A new biosensor for the direct detection of organophosphorus (OP) neurotoxins has been developed utilizing cryoimmobilized, recombinant E. coli cells capable of hydrolyzing a wide spectrum of OP pesticides and chemical warfare agents. The biological transducer was provided by the enzymatic hydrolysis of OP neurotoxins by organophosphate hydrolase which generates two protons through a reaction in which P-O, P-F, P-S or P-CN bonds are cleaved, and the proton release corresponded with the quantity of organophosphate hydrolyzed. This stoichiometric relationship permitted the creation of a potentiometric biosensor for detection of OP neurotoxins and a pH-based assay was developed as a direct function of the concentration of OP neurotoxins and the immobilized biomass. In these studies utilizing paraoxon as the substrate, neurotoxin concentration was determined with two different types of measuring units containing immobilized cells: (1) a stirred batch reactor; and (2) a flow-through column minireactor. A pH glass electrode was used as the physical transducer. The linear detection range for paraoxon spanned a concentration range of 0.25-250 ppm (0.001-1.0 mM). The response times were 10 min for the batch reactors and 20 min for the flow-through systems. It was possible to use the same biocatalyst repetitively for 25 analyses with a 10 min intermediate washing of the biocatalyst required for reestablishing the starting conditions. The cryoimmobilized E. coli cells exhibited stable hydrolytic activity for over 2 months under storage in 50 mM potassiumphosphate buffer at +4 degrees C and provide the potential for the development of a stable biotransducer for detecting various OP neurotoxins.

Biosensing Techniques↗
PubMed2001

Direct detection of 16S rRNA in soil extracts by using oligonucleotide microarrays.

We report on the development and validation of a simple microarray method for the direct detection of intact 16S rRNA from unpurified soil extracts. Total RNAs from Geobacter chapellei and Desulfovibrio desulfuricans were hybridized to an oligonucleotide array consisting of universal and species-specific 16S rRNA probes. PCR-amplified products from Geobacter and Desulfovibrio were easily and specifically detected under a range of hybridization times, temperatures, and buffers. However, reproducible, specific hybridization and detection of intact rRNA could be accomplished only by using a chaperone-detector probe strategy. With this knowledge, assay conditions were developed for rRNA detection using a 2-h hybridization time at room temperature. Hybridization specificity and signal intensity were enhanced using fragmented RNA. Formamide was required in the hybridization buffer in order to achieve species-specific detection of intact rRNA. With the chaperone detection strategy, we were able to specifically hybridize and detect G. chapellei 16S rRNA directly from a total-RNA soil extract, without further purification or removal of soluble soil constituents. The detection sensitivity for G. chapellei 16S rRNA in soil extracts was at least 0.5 microg of total RNA, representing approximately 7.5 x 10(6) Geobacter cell equivalents of RNA. These results suggest that it is now possible to apply microarray technology to the direct detection of microorganisms in environmental samples, without using PCR.

Base Sequence↗
PubMed1996

Substances interfering with direct detection of Mycobacterium tuberculosis in clinical specimens by PCR: effects of bovine serum albumin.

Interfering substances have been reported to inhibit PCR assays for the direct detection of Mycobacterium tuberculosis in clinical specimens. Using an internal control, we determined that 52% of respiratory specimens interfered with our PCR assay. On the basis of these findings, we tried to circumvent the problem by simply diluting prepared sediments. With sediment from a routinely processed sputum known to be inhibitory to PCR, one aliquot was prepared in a routine manner for PCR. Remaining sediment was diluted in phosphate-buffered saline, Middlebrook 7H10 broth, or BACTEC 12B broth; an internal control was added to all reaction mixtures and controls. Internal control was detected only in the sample diluted with BACTEC 12B medium. Components of the BACTEC 12B medium including PANTA reagent (polymyxin B, amphotericin B, nalidixic acid, trimethoprim, and azlocillin), reconstituting fluid, 0.2% glycerol, 0.05% Tween 80, and 0.05% bovine serum albumin (BSA) were tested in a similar manner. Only 0.05% BSA resulted in amplification of the internal control DNA. Varying concentrations of BSA were added to 11 aliquots of a respiratory sediment known to be inhibitory to the PCR. Internal control was detected in all reaction mixtures containing 0.00038 to 0.1% BSA. To determine the ability of BSA to override inhibition, respiratory specimens were run in triplicate: undiluted, diluted 1:2 with BACTEC 12B medium, or diluted with 0.026% BSA. For 21 of 22 inhibitory specimens, BSA was able to override the presence of interfering substances. These data suggest that the presence of BSA in a PCR assay is critical for the direct detection of M. tuberculosis in respiratory specimens.

Animals↗
PubMed1993

Stability of respiratory syncytial virus antigen due to buffer treatment for direct detection in nasopharyngeal specimens with enzyme immunoassay.

We developed an enzyme immunoassay (direct EIA; Enzygnost RSV[Ag]) for the direct detection of respiratory syncytial virus (RSV) antigen in nasopharyngeal specimens (NPS). The test procedure is the same as our recently described direct EIA for detection of influenza A and B virus antigens in NPS. For practical purposes it is of advantage to differentiate respiratory viruses on the same microtitration plate in the same run. The test shows no limitations by sample consistency, and results are obtained within 4 hr. In contrast to other test systems, sonification is not necessary. This is due to the sample buffer STD. We studied the influence of sample buffer STD on the stability of RSV (strain Long) antigen at different temperatures over a period of 7 days. PBS-BSA-buffer served as control. The treatment and storage of RSV (strain Long) with sample buffer STD at room temperature or at 4 degrees C showed no decrease of antigen detectability. The antigen is very stable in contrast to the storage of RSV (strain Long) in PBS-BSA buffer during the observation period of 7 days. Consequently, when NPS are stored in sample buffer STD, results of direct EIA are independent from the time of transport and temperature within 7 days. Thirty-eight NPS from infants with confirmed RSV infection were investigated. Confirmation was performed by virus isolation (n = 29) or with commercially available enzyme immunoassays or immunofluorescence test (n = 9). The direct EIA showed a specificity of 99.3% (n = 140) and a sensitivity of 95% (n = 38).

Antigens, Viral↗
PubMed1996

Detecting directional hypokinesia: the epidiascope technique.

A new technique to detect directional hypokinesia is proposed.. Subjects with unilateral neglect were administered cancellation tests with an epidiascope used to dissociate visual input from motor output. Two different cancellation tasks were performed in order to compare this technique with the method devised by Tegner and Levander [Brain 114, 1943-1951, 1991]. No difference in detecting hypokinetic deficits was observed; however, our method was easier to perform and more suitable for clinical testing.

Aged↗
PubMed2005

Direct detection of Mycobacterium tuberculosis complex in pulmonary and extrapulmonary samples by BDProbeTec ET system.

BDProbeTec ET (Becton Dickinson, Sparks, Md, USA) is a fully automated walkaway system based on strand displacement amplification (SDA) technology that provides a method for the direct detection of Mycobacterium tuberculosis complex (MTBC) target sequence. The purpose of this study was to evaluate the ability of BDProbeTec ET system to detect MTBC directly from clinical specimens and compare the results with staining and culture. From February 2002 through December 2003 a total of 1521 [pulmonary (n=1329) and extrapulmonary (n=192)] specimens from 1518 patients were examined by BDProbeTec ET system for the detection of MTBC and the results were compared to those obtained by microscopy and liquid culture (BACTEC 9000 MB, Becton Dickinson). MTBC was cultivated from 65 specimens (60 pulmonary and 5 extrapulmonary) of which 43 (66.2%) (42 pulmonary and 1 extrapulmonary) were smear positive and 22 (33.8%) (18 pulmonary and 4 extrapulmonary) were smear negative. BDProbeTec ET detected MTBC in 58 (55 pulmonary and 3 extrapulmonary) of the 65 culture-positive specimens. Although the BDProbeTec ET system gave five false-negative results among the 18 smear-negative culture-positive pulmonary specimens, our results demonstrate that the BDProbeTec ET system is a reliable tool in smear-positive samples and given its technical characteristics it can be used for the rapid detection of MTBC in either pulmonary or extrapulmonary samples.

Bronchoalveolar Lavage Fluid↗
PubMed2006

Evaluation of immunomagnetic separation-polymerase chain reaction in direct detection of Brucella abortus and Brucella melitensis from cheese samples.

The current study was carried out to assess the use of immunomagnetic separation-polymerase chain reaction (IMS-PCR) in direct detection of Brucella abortus and B. melitensis from soft cheese and to examine a relatively small number of field samples for the presence of these species. Two methodologies, one with IMS and the other without IMS, were employed for recovery of the Brucella species from cheese samples. IMS in conjunction with the PCR assay was determined to detect as low as 3x10(2) bacteria/mL, while the limit of detection with the other extraction procedure was 3x10(3) bacteria/mL. In the analysis of 40 cheese samples collected from various markets, only B. abortus was detected by PCR using both DNA extraction procedures in two (5%) samples. No positive results were obtained by culture and B. melitensis was not found in any cheese samples examined. The results suggest that this technique is promising owing to its pace and high sensitivity and should aid in direct detection of Brucella species from complex food samples.

Brucella abortus↗
PubMed2005

13C direct detected experiments: optimization for paramagnetic signals.

To optimize 13C direct detected experiments for the observation of signals close to a paramagnetic center, we have assessed the sensitivity of different sequences based on CO-Cali coherence transfer. Features of CACO experiments were tested for Calbindin D9k, in which one of the two native Ca2+ ions is replaced by the paramagnetic Ce3+ ion. We have studied the comparison of single vs multiple quantum coherence transfer evolution as well as the influence of in-phase vs anti-phase detection of 13CO signals and finally the comparison of a coherence transfer step based on a CyO in plane with respect to a Cy ali in plane. The acquisition of the anti-phase component of the signal, accomplished by the removal of the last refocusing steps, allowed the identification of some signals unobserved with other pathways. The structural dependency of paramagnetism-induced nuclear relaxation is such that the identification of the most suitable coherence transfer pathway is not known "a priori" but it is driven by the relative proximity of Cali and CO to the paramagnetic center.

Animals↗
PubMed1988

Direct detection of paramagnetic species in adriamycin perfused rat hearts.

Direct detection of paramagnetic species in control and adriamycin-perfused rat hearts has been carried out. Depending on the flow rate of the perfusion solution (8,4,2 and 1 ml/min) different paramagnetic species were observed: Fe(III)(g = 2.12) at 4 ml/min; three types of oxygen centered radicals of which two in control hearts (g = 2.05 g = 2.038 g = g = 2.008) and the third one (g = 2.03 g = 2.005) in adriamycin perfused hearts, at 2 ml/min. The latter radical was the only one observed at perfusion rate of 1 ml/min both in control and adriamycin treated systems. A relationship between the intracellular enzymatic reductive activation of the anthracycline and the occurrence of ischemic conditions (4,2 and 1 ml/min) in myocardial tissues is proposed basing on the relative amounts of the paramagnetic species above mentioned.

Animals↗
PubMed1999

Multicenter evaluation of two commercial amplification kits (Amplicor, Roche and LCx, Abbott) for direct detection of Mycobacterium tuberculosis in pulmonary and extrapulmonary specimens.

Direct detection of Mycobacterium tuberculosis was performed in parallel with the Amplicor M. tuberculosis test (Roche Diagnostic System, USA) and the LCx M. tuberculosis (Abbott Diagnostic Division, USA) on 697 samples, collected from 481 patients, in three different Italian laboratories. Though both systems are licensed only for pulmonary specimens, 113 extrapulmonary specimens (represented mainly by pleural fluids, cerebrospinal fluids and urines) were included in the study. Amplification results were compared with acid-fast microscopy, culture, and identification of isolates. Final clinical diagnosis was used to resolve discrepant results. M. tuberculosis was detected in 105 specimens by both assays, whereas 561 were agreeing negatives; 21 and 6 of the remaining true-positive samples scored positive with LCx only and with Amplicor only, respectively. There were three false-positives with LCx and one false-positive with Amplicor. The diagnostic sensitivity of both methods was significantly better when only respiratory specimens were considered (78% versus 59% in nonrespiratory samples with Amplicor, and 88% versus 65% with LCx). Our data reveal a significantly better sensitivity of the LCx (p = 0.026) and a slight better specificity of the Amplicor assay. It is noteworthy that 16 of the 21 Amplicor-negative specimens in which LCx detected M. tuberculosis were culture negative, thus suggesting that the higher diagnostic sensitivity of the latter assay is attributable to its better analytical sensitivity. However, the majority of such samples originated from patients under antimicrobial treatment, which makes uncertain the clinical significance of such increased sensitivity. Considering true-positive for LCx and true-negative for Amplicor, the 16 culture-negative/LCx-positive/Amplicor-negative specimens resulted true-positives after the resolution of discrepancies, the final overall sensitivity and specificity values of the LCx assay were not significantly different from the ones of the Amplicor assay.

Culture Media↗
PubMed1993

Direct detection of PCR products for HLA class II typing.

Direct detection of the PCR, or DD-PCR is proposed as an efficient method for performing PCR assays. Following the PCR reaction, ethidium homodimer dye is added to the reaction mixture and read by fluorescence. The dye step circumvents the necessity of running reactions on agarose gel electrophoresis, which is the current standard. This simple modification should find wide application for assays utilizing the PCR reaction. Here we show the ready detection of HLA class II polymorphism.

Base Sequence↗
PubMed1997

Evaluation of a commercial ligase chain reaction kit (Abbott LCx) for direct detection of Mycobacterium tuberculosis in pulmonary and extrapulmonary specimens.

Direct detection of Mycobacterium tuberculosis by means of a commercial ligase chain reaction DNA amplification method (LCx M. tuberculosis; Abbott Diagnostics Division, Abbott Park, Ill.) was investigated with 511 (including 147 extrarespiratory) specimens collected from 358 patients. LCx results were compared with standard microbiological data, and conflicting cases were resolved according to the final clinical diagnosis. M. tuberculosis was detected in 45 of 358 subjects by means of the LCx test. The test was negative for all 30 specimens with mycobacteria other than M. tuberculosis. The sensitivity, specificity, and positive and negative predictive values for the LCx test, compared with culture results, were 93.90, 92.31, 70.00, and 98.75%, respectively; these values rose in resolved cases to 95.53, 99.25, 97.27, and 98.75%, respectively. With respiratory specimens, for which the LCx system is licensed, the sensitivity reached 98.97%. In patients with a final clinical diagnosis of tuberculosis the sensitivity of the LCx system was 89.36% compared to 82.98% for cultures and 78.72% for microscopy. We conclude that the LCx test is user friendly, rapid, fairly sensitive, and highly specific. It can also be effectively used on extrapulmonary specimens provided possible false-negative results are taken into account. However, the use of LCx test appears to be less appropriate for the monitoring of antituberculosis therapy, as the majority of samples from treated tuberculosis patients gave consistently positive results, despite the sterilization of cultures.

Bacteriological Techniques↗
PubMed1995

Evaluation and validation of a monoclonal immunofluorescent reagent for direct detection of Bordetella pertussis.

An outbreak of pertussis in Manitoba, Canada, provided an opportunity to evaluate the recently developed monoclonal antibody (MAb) BL-5 for the direct detection of Bordetella pertussis. The MAb recognizes a lipooligosaccharide epitope. A total of 1,507 consecutive nasopharyngeal swabs for culture and companion smears for direct fluorescent-antibody (DFA) detection were evaluated at Cadham Provincial Laboratory between September and November 1994. The cutoff for DFA positivity was four fluorescing organisms with morphology characteristic of B. pertussis. PCR analysis for B. pertussis DNA was performed on a subset of 100 smears by eluting material from the slides after DFA examination. In comparison with culture, the sensitivity, specificity, and positive and negative predictive values of BL-5 were 65.1% (41 of 63 samples), 99.6% (1,438 of 1,444 samples), 87.2% (41 of 47 samples), and 98.5% (1,438 of 1,460 samples), respectively. The sensitivity of culture compared with PCR was 45.5% (10 of 22 samples) for the subset of 100 specimens tested by both procedures. An expanded "gold standard" of positivity by culture or PCR for these 100 specimens resulted in DFA sensitivity, specificity, and positive and negative predictive values of 32.3, 97.1, 83.3, and 76.1%, respectively. The utility of MAb BL-5 for direct detection of B. pertussis in a clinical laboratory setting has been demonstrated by this investigation.

Adult↗
PubMed1992

Direct detection of bovine leukemia virus infection: practical applicability of a double polymerase chain reaction.

A double polymerase chain reaction (PCR) assay has been devised for the direct detection of bovine leukemia virus (BLV). The assay was directly performed on blood leukocytes, avoiding the DNA-purification procedures. The PCR products were identified by gel-electrophoresis and the specificity of the test was confirmed by hybridization with a biotinylated oligonucleotide probe. When testing the sensitivity of PCR, less than eight genome copies of the provirus were detected in the background of two million negative lymphocytes. In a BLV infected herd 22 animals of various age groups were examined by the indirect (serological) diagnostic tests of agar-gel immunodiffusion and indirect ELISA as well as by the direct detection method of PCR. The tests were repeated at monthly intervals on five occasions. When examining the specimens from cows and heifers, a close agreement was found between the results of the various methods. The newborn calves, which were the offspring of BLV infected mothers, were consequently negative in PCR throughout the experimental period. However, in the indirect tests the calves were positive during the first samplings and became negative only around four months of age. Since the indirect tests can not discriminate infection from colostral immunity, PCR proved to be a useful complementary assay for the safe diagnosis of BLV infection in young calves.

Animals↗
PubMed1992

The magnetic immuno polymerase chain reaction assay for direct detection of salmonellae in fecal samples.

Direct polymerase chain reaction (PCR)-based detection with fecal specimens is hampered by inhibitory compounds, such as bilirubin and bile salts. These fecal compounds showed significant inhibition of PCR at low concentrations (10 to 50 micrograms/ml). For direct PCR analysis, fecal samples must be diluted 500-fold to overcome inhibition. Therefore, the magnetic immuno PCR assay (MIPA), which combines immunomagnetic separation by using specific monoclonal antibodies and PCR, was used to directly detect salmonellae in feces from humans. Immunomagnetically extracted stool samples needed to be diluted only 10-fold when 1 microgram of T4 gene 32 protein was added to the PCR. The MIPA sensitivity obtained was 10(5) CFU/ml of feces. A panel of monoclonal antibodies specific for Salmonella serogroups A to E was used to extract salmonellae from clinical samples. MIPA detection of salmonellae occurred with 11 out of 14 stool samples stored at 4 degrees C for 2 months. MIPA detection of salmonellae in stool samples is a promising, fast method for detection and identification.

Antibodies, Monoclonal↗
PubMed2006

Direct detection of methicillin-resistant Staphylococcus aureus in clinical specimens by a nucleic acid-based hybridisation assay.

The occurrence of methicillin-resistant Staphylococcus aureus (MRSA) is still increasing worldwide and is associated with significant morbidity, mortality and hospital costs. Screening for MRSA plays a key role in limiting further nosocomial spread of this organism. Control measures require a rapid and sensitive test for direct detection of MRSA carriage. This study evaluated an easy-to-use PCR-hybridisation assay for the direct detection of MRSA in clinical swab specimens. In total, 508 pairs of swabs from 242 patients at risk for MRSA carriage were analysed by the standard culture method and the PCR assay. One swab was used for PCR and culture, while the second was used for culture only. Of the 508 pairs tested, 37 were positive by culture and 35 were positive by PCR. Among the 471 culture-negative specimens, 465 were negative by PCR and six were PCR-positive. The PCR assay had a sensitivity of 94.59%, a specificity of 98.73%, a positive predictive value of 85.37%, and a negative predictive value of 99.57%. The PCR-hybridisation assay enabled reliable detection of MRSA carriage in c. 4 h, thereby allowing its effective use in an MRSA control strategy.

Bacteriological Techniques↗
PubMed1996

Comparison of the sensitivity & specificity of a polyclonal versus monoclonal capture antibody based Bead ELISA for direct detection of cholera toxin from stool specimens.

This study was conducted to examine whether substitution of polyclonal anti-CT IgG (PAb) coated beads with monoclonal anti-CT IgG, (MAb) coated beads would enhance the sensitivity and specificity of the Bead ELISA for direct detection of CT in stool samples of cholera patients. Sensitivity of MAb Bead ELISA was found to be more efficient (92.7%) than the PAb Bead ELISA (88.2%) in comparison to the 'gold standard' method of conventional culture method (CM). However, the MAb based Bead ELISA had lower specificity (45%) than that of PAb Bead ELISA (51.8%). Further, the positive predictive value was also lower in MAb Bead ELISA (69.9%) as compared to PAb Bead ELISA (82.1%). Generally, all the statistical evaluation demonstrated better agreement between PAb (77.9%) and MAb (72.6%) Bead ELISAs in direct detection of free CT and culture method in confirming the causative organism i.e., Vibrio cholerae in stool specimens. When the two assay systems, viz., PAb and MAb Bead ELISAs were compared, the superiority of the PAb Bead ELISA over MAb Bead ELISA was clearly evident as it had 82.4 per cent of agreement value.

Antibodies, Monoclonal↗