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At least 595 records · Page 33Linked to original sources
PubMed2006

Development and evaluation of triplex PCR for direct detection of mycobacteria in respiratory specimens.

AIMS: To develop a method for the simultaneous detection of Mycobacterium tuberculosis, Mycobacterium avium-intracellularecomplex (MAC), and other mycobacteria in clinical specimens using triplex PCR. METHODS AND RESULTS: The target of amplification was the internal transcribed spacer region between the l6S and 23S rDNA genes. Twenty-two mycobacterial type strains, 118 M. tuberculosis, 87 other mycobacteria, 75 nonmycobacterial pathogens, 115 respiratory specimens from confirmed cases of tuberculous or other mycobacterial diseases, and sputa from 50 patients with nonmycobacterial respiratory diseases were tested. In M. tuberculosis, 322 bp pan-mycobacterial and 133 bp species-specific bands were observed. In MAC, the respective bands were 322 and 84 bp. The other mycobacteria showed single pan-mycobacterial bands of approx. 300-350 bp. Nonspecific amplicons were not found in any of the nonmycobacterial pathogens. In the tuberculosis specimens, 96.4% of smear-positive specimens and 70.2% of smear-negative specimens showed positive reactions. Specimens from two patients with MAC infection were MAC positive. Only 1 of 50 specimens from nonmycobacterial diseases was positive (2.0%). CONCLUSION, SIGNIFICANCE AND IMPACT OF THE STUDY: Triplex PCR enables accurate and rapid diagnosis of tuberculosis and probably is useful for the detection of MAC and other mycobacteria in respiratory specimens.

Bronchoalveolar Lavage Fluid↗
PubMed2006

Linking bacteriophage infection to quorum sensing signalling and bioluminescent bioreporter monitoring for direct detection of bacterial agents.

AIM: To incorporate into the lambda phage genome, a luxI-based acyl-homoserine lactone (AHL) synthase genetic construct and exploit the autoamplified power of quorum sensing to translate a phage infection event into a chemical signature detectable by a lux-based bioluminescent bioreporter, with focus towards facile detection of microbial pathogens. METHODS AND RESULTS: The luxI gene from Vibrio fischeri was inserted into the lambda phage genome to construct a model phage-based biosensor system for the general detection of Escherichia coli. The AHL signalling molecules synthesized upon phage infection are detected by an AHL-specific bioluminescent bioreporter based on the luxCDABE gene cassette of V. fischeri. The assay generates target-specific visible light signals with no requisite addition of extraneous substrate. This binary reporter system was able to autonomously respond to lambda phage infection events at target E. coli concentrations ranging from 1 x 10(8) to 1 CFU ml(-1) within 1.5-10.3 h, respectively, in pure culture. When assayed against artificially contaminated lettuce leaf washings, detection within an E. coli inoculum range from 1 x 10(8) to 130 CFU ml(-1) was achieved within 2.6-22.4 h, respectively. CONCLUSIONS: The initial feasibility of binary phage-based reporter assays indicates that quorum sensing can be used to translate a phage infection event into an autoamplified chemical signature. SIGNIFICANCE AND IMPACT OF STUDY: With further modification, binary phage-based reporter assays may be capable of rapidly and cost effectively detecting pathogenic agents at very low population densities.

4-Butyrolactone↗
PubMed1993

Prenatal diagnosis of fragile X syndrome by direct detection of the dynamic mutation due to an unstable DNA sequence.

The fragile X syndrome is the most common familial form of mental retardation. The mutation causing the syndrome is dynamic mutation due to an unstable DNA (CCG)n repeat localized at Xq27.3. We have previously reported a PCR procedure to prepare a diagnostic probe, pPCRfx1, which can be used to determine the genotype of fragile X mutation individuals by Southern blot analysis. In the present study, pPCRfx1 was applied to the prenatal diagnosis, using chorionic villus cells, of a fetus which was at risk of having fragile X syndrome. In the PstI assay, the Southern blot showed the typical pattern of a female carrier with the full mutation. Analysis of the DNA methylation patterns by EcoRI + EagI assay showed that the EagI restriction site was not methylated on the mutated X chromosome of chorionic villi, but the sites were totally methylated in the brain and other tissues of the fetus. Thus the fetus was diagnosed to be a heterozygous female carrier of the dynamic mutation involved in the fragile X syndrome.

Blotting, Southern↗
PubMed2005

Direct detection of reactive oxygen species ex vivo.

Oxidative stress is thought to play an important role in the initiation and progression of renal, cardiovascular, neoplastic, and neurodegenerative diseases. It is also widely believed that oxidative stress is a main cause of aging. Although considerable progress has been made in the understanding of the sources and actions of oxidative stress, the true role of oxygen-derived free radicals in the pathology of most human diseases largely remains to be determined. One major obstacle for radical research is the lack of specific and sensitive methods to quantify oxidative stress in vivo and in vitro. Although a multitude of different assays is available to assess free radical generation, each of these methods has substantial limitations. This article will provide a brief review on the most frequently used techniques to assess oxygen-derived free radical generation in isolated tissue preparations and cells. Emphasis will be put on most recent technical innovations and the shortcomings associated with current techniques.

Animals↗
PubMed2006

[Evaluation of the new nucleic acid amplification system for direct detection of Chlamydia trachomatis and Neisseria gonorrhoeae in women].

The performance of a real-time DNA amplification assay, BD ProbeTec ET System (BDPT, BD Diagnostic Systems), to detect Chlamydia trachomatis and Neisseria gonorrhoeae on endocervical and oropharyngeal samples was evaluated. After obtaining informed consent, 364 endocervical, 363 urine and 247 oropharyngeal specimens were collected from 307 cases. The overall agreement rate of the BDPT and Amplicor (AMP, Roche) assays for the detection of C. trachomatis and N. gonorrhoeae in endocervical samples was 99.2% (361/364) for C. trachomatis and 99.5% (362/364) for N. gonorrhoeae. Assay of oropharyngeal swabs by the BDPT yielded 21 C. trachomatis positives, and 19 of them were C. trachomatis negative by the DNA probe assay (Gen-Probe PACE). The AMP assay showed that 16/19 (84.2%) of the BDPT +/DNA probe - samples were positive. The BDPT also yielded 21 N. gonorrhoeae positives, 15 of which were negative with the DNA probe. Additional testing showed that all 15 BDPT +/DNA probe - samples were positive by the established nested PCR method. Our data suggest that the performance of the BDPT is comparable to that of AMP for detection of C. trachomatis and N. gonorrhoeae in endocervical swab samples and that it may be a useful method for detecting of C. trachomatis and N. gonorrhoeae in oropharyngeal samples clinically.

Cervix Uteri↗
PubMed1998

Direct detection of viable bacteria, molds, and yeasts by reverse transcriptase PCR in contaminated milk samples after heat treatment.

A fast, sensitive, and target contaminant-modulable method was developed to detect viable bacteria, molds, and yeasts after heat treatment. By reverse transcriptase PCR with elongation factor gene (EF-Tu or EF-1 alpha)-specific primers, the detection level was 10 cells ml of milk-1. The simplicity and rapidity (4 h) of the procedure suggests that this method may be easily transposable to other foods and other contaminants.

Animals↗
PubMed2001

Direct detection by in situ PCR of the amoA gene in biofilm resulting from a nitrogen removal process.

Ammonia oxidation is a rate-limiting step in the biological removal of nitrogen from wastewater. Analysis of microbial communities possessing the amoA gene, which is a small subunit of the gene encoding ammonia monooxygenase, is important for controlling nitrogen removal. In this study, the amoA gene present in Nitrosomonas europaea cells in a pure culture and biofilms in a nitrifying reactor was amplified by in situ PCR. In this procedure, fixed cells were permeabilized with lysozyme and subjected to seminested PCR with a digoxigenin-labeled primer. Then, the amplicon was detected with an alkaline phosphatase-labeled antidigoxigenin antibody and HNPP (2-hydroxy-3-naphthoic acid-2'-phenylanilide phosphate), which was combined with Fast Red TR, and with an Alexa Fluor 488-labeled antidigoxigenin antibody. The amoA gene in the biofilms was detected with an unavoidable nonspecific signal when the former method was used for detection. On the other hand, the amoA gene in the biofilms was detected without a nonspecific signal, and the cells possessing the amoA gene were clearly observed near the surface of the biofilm when Alexa Fluor 488-labeled antidigoxigenin antibody was used for detection. Although functional gene expression was not detected in this study, detection of cells in a biofilm based on their function was demonstrated.

Biofilms↗
PubMed1982

Enzyme immunoassay for direct detection of influenza type A and adenovirus antigens in clinical specimens.

Detection of viral antigens in specimens without prior cultivation in cell culture provides the most rapid method for specific viral diagnosis. A solid-phase, double-antibody enzyme immunoassay was developed for this purpose and tested with clinical specimens containing influenza type A and adenovirus. Polystyrene microtiter wells were the solid phase and were coated with virus-specific guinea pig immunoglobulins. Specimens were added, and bound viral antigens were detected by addition of virus-specific rabbit immunoglobulins followed by enzyme-labeled goat antirabbit immunoglobulin G. Two methods of labeling goat anti-rabbit immunoglobulin G with horseradish peroxidase were investigated: covalent attachment and a noncovalent, immunological binding of antibody to enzyme, the peroxidase-antiperoxidase method. Both methods of labeling resulted in assays that could detect 10(3.5) 50% tissue culture infectious doses of influenza type A and 10(3.8) 50% tissue culture infectious doses of adenovirus. Equal sensitivity was noted with alkaline phosphatase-labeled goat anti-rabbit immunoglobulin G. An increase in sensitivity of three- to sixfold was achieved when virus-specific rabbit immunoglobulins and conjugate were diluted in 1% gelatin. The solid-phase, double-antibody enzyme immunoassay detected influenza type A and adenovirus in isolation-positive clinical specimens with 53% (21/40) and 62% (13/21) efficiency, respectively. The solid-phase, double-antibody enzyme immunoassay has considerable potential as a practical and rapid method for detection of respiratory viral antigens in nasal wash and throat swab specimens. For optimal value, however, greater sensitivity than was provided by the present methods is desirable.

Adenoviridae↗
PubMed1982

Rapid diagnosis of rotavirus infection by direct detection of viral nucleic acid in silver-stained polyacrylamide gels.

A rapid simple technique for the diagnosis of rotavirus has been developed based on the sensitive detection of rotavirus double-stranded RNA genome segments separated in polyacrylamide gels. The method utilizes a recently described ultrasensitive silver stain for polypeptides, which can also detect subnanogram amounts of nucleic acid. The sensitivity of the technique is comparable with that of electron microscopy or enzyme-linked immunosorbent assay.

Animals↗
PubMed1986

Direct detection and differentiation of fastidious and nonfastidious adenoviruses in stools by using a specific nonradioactive probe.

Restriction fragments of fastidious human adenovirus type 41 (Ad41) were cloned in vector plasmid pBR322. A rapid and sensitive nonradioactive molecular-hybridization technique (M. Renz and C. Kurz, Nucleic Acids Res. 12:3435-3444, 1984) showed that one clone specifically detected fastidious Ad40 and Ad41 (subgenus F) without cross-hybridization with nonfastidious adenoviruses. This clone was mapped in a region of the Ad41 genome corresponding to early transcription unit E1B of Ad2. A number of DNAs from fastidious and nonfastidious adenoviruses were extracted, without cultivation, from stools of children with gastroenteritis and were hybridized with an Ad2 probe and with the cloned probe, allowing the differentiation of the two groups of viruses. This method could detect DNA quantities as low as 10 pg and should be particularly suitable for stool samples containing adenoviral DNA in amounts too low to be detected by staining with ethidium bromide.

Adenovirus Infections, Human↗
PubMed1987

Monoclonal antibody passive hemagglutination and capture enzyme-linked immunosorbent assays for direct detection and quantitation of F41 and K99 fimbrial antigens in enterotoxigenic Escherichia coli.

Production of diarrhea in neonatal calves by enterotoxigenic Escherichia coli depends on its ability to attach to the epithelial cells of the intestine via surface adhesins called pili or fimbriae and to secrete enterotoxins. The most important of these fimbriae are designated K99 and F41. We produced and characterized a murine monoclonal antibody specific to F41. This monoclonal antibody and a K99-specific monoclonal antibody were used to develop sensitive and specific passive hemagglutination and capture enzyme-linked immunosorbent assays (ELISAs) for detection and quantitation of F41 and K99 antigens in E. coli cultures and culture supernatants. The capture ELISA systems exhibited excellent sensitivity and specificity, whereas the passive hemagglutination systems appeared to be oversensitive. The ability of the capture ELISAs to detect K99 and F41 fimbrial antigens in fecal specimens from calves was evaluated. Fimbrial antigens were detected in six of six specimens from scouring calves but not in four of four specimens from nonscouring calves.

Animals↗
PubMed1989

Direct detection of cytomegalovirus from bronchoalveolar lavage samples by using a rapid in situ DNA hybridization assay.

An in situ DNA hybridization assay was compared with centrifugation culture for rapid detection of cytomegalovirus (CMV) from bronchoalveolar lavage (BAL) samples. Eighty BAL samples were inoculated into both centrifugation culture and standard culture. Cytospin preparations of the BAL samples were studied in a 75-min in situ DNA hybridization assay using the PathoGene CMV kit (Enzo Biochem, Inc., New York, N.Y.). Of the 80 samples, 39 (49%) were positive for CMV; 37 of 39 (95%) were positive by centrifugation culture, 34 of 39 (87%) were positive in standard culture, 24 of 39 (62%) were positive by in situ hybridization, and 20 of 39 (56%) were positive by histologic and/or immunofluorescence techniques. The in situ hybridization assay detected 23 of the 37 samples positive in centrifugation culture, for a sensitivity of 62% and a specificity of 98%. We conclude that the in situ hybridization assay is a specific and more rapid test than centrifugation culture and standard culture for diagnosis of CMV pulmonary infection. For the clinical laboratory, however, current hybridization methods are not sufficiently sensitive to replace centrifugation culture for detection of CMV in BAL specimens.

Bronchoalveolar Lavage Fluid↗
PubMed1990

Direct detection of Mycobacterium tuberculosis in clinical specimens by DNA amplification.

A polymerase chain reaction assay was developed for the amplification of a 336-bp repetitive fragment in the chromosome of Mycobacterium tuberculosis. The assay is specific for M. tuberculosis and can be used to detect the amount of DNA present in less than 10 organisms. It was used to demonstrate M. tuberculosis DNA in 14 of 26 clinical specimens (from cerebrospinal fluids, pleural fluid aspirates and biopsies, pericardial fluid aspirates, and an open lung biopsy), and it was shown to be at least as sensitive as conventional culture techniques with these specimens.

Base Sequence↗
PubMed1992

Evaluation of an enzyme-linked immunoassay employing a covalently bound capture antibody for direct detection of herpes simplex virus.

The FDL enzyme-linked immunosorbent assay (ELISA; Fairleigh Dickinson Laboratories, Inc., Abilene, Tex.) for the detection of herpes simplex virus (HSV) makes use of a covalently attached antibody. This assay was compared with viral isolation and with the Ortho HSV Antigen Detection ELISA (Ortho Diagnostic Systems, Inc., Raritan, N.J.). One hundred forty-eight specimens were tested. The FDL ELISA identified 66 of 104 specimens from which HSV was isolated, yielding a sensitivity of 63% and a specificity of 95%. These results compared favorably with those obtained by using the Ortho ELISA. The total test time was shorter and the washing step was simpler than that with the Ortho assay, making the FDL assay an attractive alternative to similar methodologies.

Antibodies, Viral↗