Browsing by Subject "PCR"

Now showing 1 - 4 of 4
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    Certification assays for HIV-1-based vectors: frequent passage of gag sequences without evidence of replication-competent viruses
    (Elsevier, 2003-11-01) Sastry, Lakshmi; Xu, Yi; Johnson, Terry; Desai, Kunal; Rissing, David; Marsh, Jonathan; Cornetta, Kenneth; Microbiology and Immunology, School of Medicine
    A principal concern regarding the safety of HIV-1-based vectors is replication-competent lentivirus (RCL). We have developed two PCR assays for detecting RCL; the first detects recombination between gag regions in the transfer vector and the packaging construct (sensitivity of detection ∼10–100 copies of target sequence). The second assay uses real-time PCR to detect vesicular stomatitis virus glycoprotein (VSVG) envelope DNA (sensitivity ∼5–50 VSVG sequences). In an attempt to amplify any RCL, test vectors were used to transduce C8166 and 293 cells, which were then screened weekly for 3 weeks. Psi–gag recombinants were routinely detected (20 of 21 analyses) in four transductions using the RRL-CMV-GFP vector. In contrast, VSVG sequences were detected only once in 21 analyses. Interestingly, p24 levels (as measured by ELISA) were occasionally detectable after 3 weeks of culture. To determine if a true RCL was present, 21-day cell-free medium was used to transduce naïve cells. No evidence of psi–gag or VSVG transfer was detected, indicating that the recombination events were insufficient to reconstitute a true RCL. These findings have important implications for the design and safety of HIV-1-based vectors intended for clinical applications.
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    Multicenter Clinical Evaluation of the Automated Aries Bordetella Assay
    (American Society for Microbiology, 2019-01-30) Relich, Ryan F.; Leber, Amy; Young, Stephen; Schutzbank, Ted; Dunn, Ronald; Farhang, Janet; Uphoff, Timothy S.; Pathology & Laboratory Medicine, IU School of Medicine
    Molecular methods offer superior sensitivity and specificity and reduce testing turnaround time from days to hours for detection of Bordetella pertussis and Bordetella parapertussis In this study, we evaluated the performance of the automated PCR-based Aries Bordetella Assay, which detects both B. pertussis and B. parapertussis directly from nasopharyngeal swab specimens. The limits of detection (LoDs) were 1,800 CFU·ml-1 for B. pertussis and 213 CFU·ml-1 for B. parapertussis The assay detected 16/18 unique B. pertussis/B. parapertussis strains. Of 71 potentially cross-reacting organisms, 5 generated false positives in 1/6 replicates; none of 6 additional Bordetella spp. were erroneously detected. Specimens were stable at 20 to 25°C for at least 10 h, at 4 to 8°C for 10 days, and at temperatures not exceeding -70°C for 6 months. Of 1,052 nasopharyngeal specimens from patients with suspected pertussis, 3.0% (n = 32) were B. pertussis positive and 0.2% (n = 2) were B. parapertussis positive. Combining these data with Aries Bordetella Assay data from 57 nasopharyngeal samples with previously confirmed B. pertussis or B. parapertussis data and with data from 50 contrived B. parapertussis samples, the proportions of positive and negative agreement of the respective Aries assays with the reference assays were 97.1% and 99.0% for B. pertussis and 100% and 99.7% for B. parapertussis The Aries Bordetella Assay provides accurate detection and distinction of B. pertussis and B. parapertussis infections within 2 h. (This study has been registered at ClinicalTrials.gov under registration no. NCT02862262.).
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    Multicenter evaluation of the BIOFIRE Joint Infection Panel for the detection of bacteria, yeast, and AMR genes in synovial fluid samples
    (American Society for Microbiology, 2023) Esteban, Jaime; Salar-Vidal, Llanos; Schmitt, Bryan H.; Waggoner, Amy; Laurent, Frédéric; Abad, Lelia; Bauer, Thomas W.; Mazariegos, Irving; Balada-Llasat, Joan-Miquel; Horn, Jared; Wolk, Donna M.; Jefferis, Alexa; Hermans, Mirjam; Verhoofstad, Irma; Butler-Wu, Susan M.; Umali-Wilcox, Minette; Murphy, Caitlin; Cabrera, Barbara; Craft, David; von Bredow, Benjamin; Leber, Amy; Everhart, Kathy; Dien Bard, Jennifer; Flores, Irvin Ibarra; Daly, Judy; Barr, Rebecca; Holmberg, Kristen; Graue, Corrin; Kensinger, Bart; Medicine, School of Medicine
    The bioMérieux BIOFIRE Joint Infection (JI) Panel is a multiplex in vitro diagnostic test for the simultaneous and rapid (~1 h) detection of 39 potential pathogens and antimicrobial resistance (AMR) genes directly from synovial fluid (SF) samples. Thirty-one species or groups of microorganisms are included in the kit, as well as several AMR genes. This study, performed to evaluate the BIOFIRE JI Panel for regulatory clearance, provides data from a multicenter evaluation of 1,544 prospectively collected residual SF samples with performance compared to standard-of-care (SOC) culture for organisms or polymerase chain reaction (PCR) and sequencing for AMR genes. The BIOFIRE JI Panel demonstrated a sensitivity of 90.9% or greater for all but six organisms and a positive percent agreement (PPA) of 100% for all AMR genes. The BIOFIRE JI Panel demonstrated a specificity of 98.5% or greater for detection of all organisms and a negative percent agreement (NPA) of 95.7% or greater for all AMR genes. The BIOFIRE JI Panel provides an improvement over SOC culture, with a substantially shorter time to result for both organisms and AMR genes with excellent sensitivity/PPA and specificity/NPA, and is anticipated to provide timely and actionable diagnostic information for joint infections in a variety of clinical scenarios.
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    Multicenter Evaluation of the Portrait Staph ID/R Blood Culture Panel for Rapid Identification of Staphylococci and Detection of the mecA Gene
    (American Society for Microbiology, 2017-04) Denys, Gerald A.; Collazo-Velez, Vanessa; Young, Stephen; Daly, Judy A.; Couturier, Marc Roger; Faron, Matthew L.; Buchan, Blake W.; Ledeboer, Nathan; Pathology and Laboratory Medicine, School of Medicine
    Bloodstream infections are a leading cause of morbidity and mortality in the United States and are associated with increased health care costs. We evaluated the Portrait Staph ID/R blood culture panel (BCP) multiplex PCR assay (Great Basin Scientific, Salt Lake City, UT) for the rapid and simultaneous identification (ID) of Staphylococcus aureus, Staphylococcus lugdunensis, and Staphylococcus species to the genus level and the detection of the mecA gene directly from a positive blood culture bottle. A total of 765 Bactec bottles demonstrating Gram-positive cocci in singles or clusters were tested during the prospective trial at 3 clinical sites. The Portrait Staph ID/R BCP results were compared with results from conventional biochemical and cefoxitin disk methods performed at an independent laboratory. Discordant ID and mecA results were resolved by rpoB gene sequencing and mecA gene sequencing, respectively. A total of 658 Staphylococcus species isolates (S. aureus, 211 isolates; S. lugdunensis, 3 isolates; and Staphylococcus spp., 444 isolates) were recovered from monomicrobial and 33 polymicrobial blood cultures. After discrepant analysis, the overall ratios of Portrait Staph ID/R BCP positive percent agreement and negative percent agreement were 99.4%/99.9% for Staphylococcus ID and 99.7%/99.2% for mecA detection.