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At least 613 records · Page 34Linked to original sources
PubMed1993

Direct detection of Escherichia coli Shiga-like toxin genes in primary fecal cultures by polymerase chain reaction.

A single pair of oligonucleotide primers was used for polymerase chain reaction amplification of a 212- or 215-bp region of Escherichia coli Shiga-like toxin (SLT) genes from crude fecal culture extracts. Genes were typed by hybridization of the polymerase chain reaction products to SLT-I- or SLT-II-specific oligonucleotide probes. The procedure was capable of detecting fewer than 10 SLT-producing E. coli organisms per ml of culture against a background of more than 10(9) other organisms per ml and provides a rapid and sensitive means of screening primary fecal cultures for the presence of such strains. When this procedure was used to test primary cultures from gut contents or feces from various patient groups, including apparently healthy infants, approximately half of all samples yielded positive results for SLT-I and/or SLT-II sequences.

Bacterial Toxins↗
PubMed1993

Direct detection of Bacillus anthracis DNA in animals by polymerase chain reaction.

Bacillus anthracis is a soil pathogen capable of causing anthrax. To establish a method for specifically detecting B. anthracis for practical applications, such as for the inspection of slaughterhouses, the cap region, which is essential for encapsulation in B. anthracis, was used in a DNA hybridization study by polymerase chain reaction (PCR). Oligonucleotide primers were designed to amplify a 288-bp DNA fragment within the capA gene by PCR. The amplified DNA sequence specifically hybridized to the DNA of B. anthracis but not to that of other bacterial strains tested. Since this PCR-based method efficiently and specifically detected the capA sequence of bacteria in blood and spleen samples of mice within 8 h after the administration of live B. anthracis, this PCR system could be used for practical applications. By using lysis methods in preparing the samples for PCR, it was possible to amplify the 288-bp DNA segment from samples containing very few bacteria, as few as only 1 sporeforming unit, indicating that the PCR detection method developed in this study will permit the monitoring of B. anthracis contamination in the environment.

Animals↗
PubMed1993

Direct detection of Mycobacterium tuberculosis in respiratory specimens in a clinical laboratory by polymerase chain reaction.

The emergence of epidemic multiple-drug-resistant (MDR) strains of Mycobacterium tuberculosis in conjunction with an increase in the number of reported cases of tuberculosis (TB) represents a major public health problem. In light of a recent outbreak of MDR M. tuberculosis at our center, we began the development of a polymerase chain reaction (PCR) assay for the rapid diagnosis of pulmonary TB using two sets of primers, one based on the IS6110 repeated sequence of M. tuberculosis and the other based on the protein antigen b (PAB). Reaction conditions were first optimized as to the appropriate extraction protocol and the concentrations of primer pairs, nucleotides, and MgCl2. Following a preliminary evaluation of the assay with clinical specimens, extraction and amplification procedures were further modified. PAB and IS6110 primers detected between 2 and 23 and 0.023 and 0.23 CFU of M. tuberculosis, respectively, in pooled, M. tuberculosis-negative sputa by our optimized PCR assay. After routine processing for mycobacteria, 734 specimens were subsequently amplified. DNA for amplification was obtained by boiling and beating the sediments with Tween 20. For each reaction, DNA (10 microliters) was added to an amplification mixture containing 12 pmol of IS6110 primers, 20 pmol of PAB primers, 2 mM MgCl2, 200 microM nucleotides, and 2.5 U of Taq polymerase and the mixture was then amplified for 40 cycles. The sensitivity and specificity of our PCR assay were 87.2 and 97.7%, respectively. We were unable to interpret the results for seven specimens (1%). In our experience, PCR proved to be a useful rapid diagnostic test for TB in a clinical setting and a valuable epidemiological tool for determining exposure groups in the hospital setting. Our findings also underscore the need for the systematic optimization of PCR assay conditions.

Antigens, Bacterial↗
PubMed1995

Evaluation of an immunoassay for direct detection of Escherichia coli O157 in stool specimens.

An enzyme-linked immunosorbent assay (ELISA) produced by LMD Laboratories, Inc., Carlsbad, Calif., was compared with culture for the detection of Escherichia coli O157. Nine of 185 stool specimens evaluated had positive results by the LMD E. coli O157 ELISA and grew E. coli O157 on culture; 174 had negative by LMD E. coli O157 ELISA results and did not grow E. coli O157 on culture. Of 174 specimens negative by LMD E. coli O157 ELISA, 117 specimens grew other enteric pathogens: Campylobacter spp. (46 isolates), Salmonella spp. (43 isolates), Yersinia spp. (20 isolates), and Shigella spp. (8 isolates). There were two indeterminant results by the LMD E. coli O157 ELISA. One stool specimen did not grow other enteric pathogens on culture, and one grew a Campylobacter sp. on culture. Both had negative LMD E. coli O157 ELISA results upon repeat testing. The LMD E. coli O157 ELISA is an accurate, easy-to-read screening method for the detection of E. coli O157 in fecal specimens.

Antibodies, Bacterial↗
PubMed1997

Specific PCR assay for direct detection of intestinal microsporidia Enterocytozoon bieneusi and Encephalitozoon intestinalis in fecal specimens from human immunodeficiency virus-infected patients.

A routine assay based on the PCR was developed for the detection of Enterocytozoon bieneusi and Encephalitozoon intestinalis in fecal samples. Two oligonucleotide primer pairs from a conserved region in the small-subunit rRNA genes of E. bieneusi (primer pair V1 and EB450) and E. intestinalis (primer pair V1 and SI500) were used to amplify microsporidian DNA. We achieved specific amplification of a 382-bp DNA fragment in E. intestinalis and a 353-bp DNA fragment in E. bieneusi. Boiling of the samples appeared to be most effective for DNA extraction. Fecal samples containing fewer than 10 microsporidia gave a positive result in the PCR assay. Fecal specimens from 30 human immunodeficiency virus-infected patients with microsporidiosis and fecal specimens from 42 patients suspected of having microsporidiosis were investigated by the PCR assay. The PCR assay was validated against standard staining methods (the Uvitex 2B and Chromotrope 2R staining methods) and immunofluorescence assay specific for E. intestinalis. This comparative study has shown that PCR improved species determination and can thus be considered a fast and reliable method for the detection and identification of each intestinal species.

AIDS-Related Opportunistic Infections↗
PubMed1998

Comparative evaluation of the new gen-probe Mycobacterium tuberculosis amplified direct test and the semiautomated abbott LCx Mycobacterium tuberculosis assay for direct detection of Mycobacterium tuberculosis complex in respiratory and extrapulmonary specimens.

Two commercial assays that detect Mycobacterium tuberculosis complex (MTB) in clinical specimens by rRNA target amplification (AMTDII) and ligase chain reaction (LCx) were evaluated. The tests were applied to 457 respiratory (n = 273) and extrapulmonary (n = 184) specimens collected from 357 patients. The results were compared with those of acid-fast staining and culture. The combination of culture and clinical diagnosis was considered to be the "gold standard." Seventy specimens were from patients with pulmonary tuberculosis and 28 specimens were from patients with extrapulmonary tuberculosis. After resolution of discrepant results, the overall sensitivities, specificities, and positive and negative predictive values for respiratory specimens were 92.8, 99.4, 98.5, and 97%, respectively, for AMTDII and 75.7, 98.8, 96.4, and 90.5%, respectively, for LCx. With extrapulmonary specimens, the overall sensitivities, specificities, and positive and negative predictive values were 78.6, 99.3, 95.6, and 96.2%, respectively, for AMTDII and 53.6, 99.3, 93.7, and 92.1%, respectively, for LCx. The level of agreement between AMTDII and LCx assay results was 78.2%. We conclude that although both nucleic acid amplification methods are rapid and specific for the detection of MTB in clinical specimens, AMTDII is significantly more sensitive than LCx with both respiratory (P = 0.005) and extrapulmonary (P = 0.048) specimens.

Bronchi↗
PubMed1999

Performance characteristics of the BDProbeTec system for direct detection of Mycobacterium tuberculosis complex in respiratory specimens.

Strand displacement amplification (SDA) technology has been established in a fully automated system known as BDProbeTec. Target sequences of the insertion sequence IS6110 and the 16S rRNA gene are simultaneously amplified, which thus allows the detection of Mycobacterium tuberculosis complex and, as an additional option, of most Mycobacterium species. Detection occurs via a chemiluminescent microwell assay that employs the simultaneous hybridization and capture of SDA products with a biotinylated capture probe and an alkaline phosphatase detector probe. We have evaluated the performance of the BDProbeTec system in detecting M. tuberculosis complex by testing 799 respiratory specimens and comparing the results to those obtained by conventional diagnostic techniques, i.e. , microscopy and culture (solid and radiometric media). M. tuberculosis was cultivated from 41 specimens, of which 28 (68.4%) were smear positive and 13 (31.6%) were smear negative. The overall sensitivity of the SDA assay was 97.6% (for smear-positive specimens, 100%; for smear-negative specimens, 92.3%), and specificity was 95. 0%. After resolution of the discrepancies by studying the patients' clinical data, sensitivity and specificity were 97.9 and 96.5%, respectively, and positive and negative predictive values were 63.9 and 99.9%, respectively. These preliminary data demonstrate that the BDProbeTec system has promising performance characteristics with respiratory specimens and that it allows the detection of M. tuberculosis complex within hours.

Adult↗
PubMed2001

Magnetic immuno-PCR assay with inhibitor removal for direct detection of Helicobacter pylori in human feces.

A PCR protocol was developed to detect Helicobacter pylori in human stool specimens. This protocol was based on the association of a magnetic immuno-PCR assay with a technique to remove inhibitors (agarose-embedded DNA preparation). Of the 47 H. pylori-positive and 57 H. pylori-negative patients included in this study, 38 were positive and 66 were negative by this new protocol. The sensitivity, specificity, and predictive values for a positive or a negative result were 80.9% (95% confidence interval [CI], 66.3 to 90.4), 100% (95% CI, 92.1 to 100), 100% (95% CI, 88.6 to 100), and 86.4% (95% CI, 75.2 to 93.2), respectively.

Adolescent↗
PubMed2002

Direct detection and characterization of Shiga toxigenic Escherichia coli by multiplex PCR for stx1, stx2, eae, ehxA, and saa.

We recently described a novel megaplasmid-encoded adhesin produced by certain Shiga toxigenic Escherichia coli (STEC) strains that lack the locus for enterocyte effacement (LEE) pathogenicity island. This adhesin, designated Saa (STEC autoagglutinating adhesin), may be a marker for a subset of LEE-negative STEC strains capable of causing severe gastrointestinal and systemic diseases in humans. In this study, we developed a pentavalent PCR assay for the detection of saa as well as other proven and putative STEC virulence genes (stx1, stx2, eae, and ehxA). The five primer pairs used in the assay do not interfere with each other and generate amplification products of 119, 180, 255, 384, and 534 bp.

Adhesins, Bacterial↗
PubMed2002

Direct detection and differentiation of Legionella spp. and Legionella pneumophila in clinical specimens by dual-color real-time PCR and melting curve analysis.

A dual-color LightCycler PCR assay targeting the 16S rDNA gene of Legionella spp. was established. By using two pairs of hybridization probes, Legionella spp. and Legionella pneumophila could be detected and differentiated simultaneously. With 26 culture-positive and 42 culture-negative respiratory specimens from patients with atypical pneumonia, 100% sensitivity and specificity was observed for L. pneumophila.

Computer Systems↗
PubMed2002

Direct detection and identification of African trypanosomes by fluorescence in situ hybridization with peptide nucleic acid probes.

We have developed a rapid and easy to perform fluorescence in situ hybridization test that allows specific identification of trypanosomes from the subgenus Trypanozoon, using peptide nucleic acid probes. Probes were designed to target subgenus-specific sequences on the multiple-copy 18S rRNA, greatly facilitating the detection of a single trypanosome.

Animals↗
PubMed2002

Direct detection of Helicobacter pylori mutations associated with macrolide resistance in gastric biopsy material taken from human immunodeficiency virus-infected subjects.

One hundred forty gastric biopsies were tested by microbiological methods and by amplifying a sequence of 23S rRNA and identifying mutations associated to clarithromycin resistance. Seventy-six specimens were positive for Helicobacter pylori. Mutational analysis revealed alterations in 18 (39.1%) of 46 and 2 (8.7%) of 23 samples from human immunodeficiency virus-seropositive and -seronegative persons, respectively. The results of the mutational analysis fully correlated with those of the susceptibility tests.

Anti-Bacterial Agents↗
PubMed2003

Real-time PCR assay targets the 23S-5S spacer for direct detection and differentiation of Legionella spp. and Legionella pneumophila.

A real-time PCR for the ABI Prism 7000 system targeting the 23S-5S spacer of Legionella spp. was developed. Simultaneous detection and differentiation of Legionella spp. and Legionella pneumophila within 90 min and without post-PCR melting-curve analysis was achieved using two TaqMan probes. In sputum samples from 23 controls and 17 patients with legionellosis, defined by positive culture, urinary antigen testing, or seroconversion, 94% sensitivity and 100% specificity were observed.

Bacterial Typing Techniques↗
PubMed2004

Direct detection of rifampin- and isoniazid-resistant Mycobacterium tuberculosis in auramine-rhodamine-positive sputum specimens by real-time PCR.

Our objective was to evaluate the feasibility of a molecular assay based on a real-time PCR technique, carried out with a LightCycler instrument (Roche Biochemicals), to identify Mycobacterium tuberculosis bacilli and to detect rifampin and isoniazid resistance in DNA extracts from sputum samples. We studied three genes: rpoB, which is associated with rifampin resistance, and katG and inhA, which are associated with isoniazid resistance. A total of 205 sputum samples collected from 108 patients diagnosed with pulmonary tuberculosis with positive auramine-rhodamine-staining (AR) sputum samples, were tested. The sensitivities of the LightCycler PCR assay for the positive AR specimens was 97.5% (200 of 205) for rpoB and inhA genes and 96.5% (198 of 205) for the katG gene. For the total number of patients tested, the sensitivity was 100% (108 of 108 patients) for rifampin, whereas the sensitivity was 98.1% (106 of 108 patients) for isoniazid. Full agreement was found with the Bactec MGIT 960 method and the genotype inferred from the LightCycler data for rifampin. The phenotypic method for isoniazid reported 13 resistant strains (> or = 0.1 microg/ml). In seven (53.8%) strains there was a concordance between both methods, but we found that six (46.2%) strains reported as resistant by the phenotypic method were determined to be susceptible by real-time PCR. For the 75 strains reported as susceptible by the phenotypic method, the concordance with the LightCycler data was 100%. Our results demonstrate that rifampin-resistant M. tuberculosis could be detected in DNA extracted from auramine-rhodamine-positive sputum samples in a single-tube assay that took less than 3 h to perform for a collection of auramine-rhodamine-positive specimens obtained from patients with culture-documented pulmonary tuberculosis. Similarly, this occurs in half of the isoniazid-resistant M. tuberculosis DNA extracted from auramine-rhodamine-positive specimens.

Antitubercular Agents↗