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Using Non-human Sourced and Banked Human Specimen Material for Nucleic Acid Based Assay Development

Using Non-human Sourced and Banked Human Specimen Material for Nucleic Acid Based Assay Development. David Norwood, Ph.D. Diagnostic Systems Division USAMRIID - Ft. Detrick. Overview. Assay design Assay optimization Purified viral RNAs Sensitivity Specificity Clinical samples

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Using Non-human Sourced and Banked Human Specimen Material for Nucleic Acid Based Assay Development

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  1. Using Non-human Sourced and Banked Human Specimen Material for Nucleic Acid Based Assay Development David Norwood, Ph.D. Diagnostic Systems Division USAMRIID - Ft. Detrick

  2. Overview • Assay design • Assay optimization • Purified viral RNAs • Sensitivity • Specificity • Clinical samples • Sensitivity • Specificity USAMRIID

  3. Assay Design • Determine Gene Target(s) • 392 bp of sequence from the RNA-directed RNA polymerase (pol) gene region (New England Journal of Medicine, 348:20 1953-1966) • Corresponds to region 15239 to 15630 of the Urbani strain. • Design specific primers and probes (Taqman) • Primer Express 2.0 for Windows • Oligo 5.0 USAMRIID

  4. Homology in pol Region USAMRIID

  5. BJ01 BJ02 BJ03 BJ04 CUHK-SU10 CUHK-W1 Frankfurt 1 GZ01 HKU-39849 HSR1 SIN2500 SIN2677 SIN2679 SIN2748 TAIWAN TAIWAN JC2003 TOR2 TW1 TWC URBANI VIETNAM ZJ01 100 % Homology in pol Region (SARS) USAMRIID

  6. Assay Optimization • Template was purified RNA from viral infected cell supernatants (Urbani strain). • 56 primer pairs tested for amplification efficiency (SYBR Green). • 13 primer pairs selected for probe testing. • 38 primer-probe sets tested for robustness. • 1 primer-probe set found superior. USAMRIID

  7. Sensitivity – Purified Viral RNA 7.5 pfu 0.75 pfu 0.075 pfu 0.0075 pfu 0.00075 pfu USAMRIID

  8. Specificity – Purified Viral RNA • Additional strains of SARS • Other related coronaviruses • None of this work done to date USAMRIID

  9. Clinical Samples • Positive clinical samples • Did not have access to positive clinical samples • Work was being done to determine if a non human primate model of disease was feasible • Negative samples (specificity) • Should be from individuals exhibiting similar clinical presentation • Should be samples from a relevant clinical matrix • Should be temporally separated from the SARS virus USAMRIID

  10. Non Human Primate Model • Three cynomologus macaques • NHP-1 and NHP-2 received an intra-nasal (IN) challenge • NHP-3 received an intravenous challenge. • Animals were monitored with telemetry implants • Strain used for challenge was Urbani USAMRIID

  11. Sample Collection / Extraction • Urine and Feces collected daily • Nasal swabs, throat swabs, rectal swabs, and blood were collected every other day beginning with day 0. • Animals were followed for 20 days post challenge • RNA was extracted from all matrices using standard Trizol extraction methods • LOD of 4x10-1 PFU/ml for Urine, Media, and Blood • LOD of 4x10-1 PFU/ml for Feces USAMRIID

  12. NHP Results • NHP-1 and NHP-2 (IN) • No disease observed by blood chemistries • NHP-2 appeared to have a mild pneumonia on day 6 which eventually resolved • NHP-3 (IV) • Developed a fever on days 5 and 6, died of a bacterial infection on day 16 (May have been from telemetry implant) • Labored breathing observed, animal was clearly sick • Left lower lobe pneumonia, resolved by the time the animal died • No disease observed by blood chemistries • PCR Results • No virus detected in feces, blood, or rectal swabs • Virus was detected in nasal swabs, throat swabs, and urine USAMRIID

  13. Throat Swabs USAMRIID

  14. Nasal Swabs USAMRIID

  15. Urine USAMRIID

  16. Negative samples • CONUS military patients • Exhibited signs of respiratory disease at time of examination • Exhibited fever at time of examination • 100 samples received, processed and tested • Throat washes • All samples were from patient examinations prior to the introduction of SARS into the US • Would other coronaviruses/infectious agents cross-react with assay? • All 100 samples were negative for by the SARS assay USAMRIID

  17. Additional work • Plan to challenge 6 more NHPs in August • 3 intra-nasal challenges and 3 intravenous challenges • Plan is to use a different strain of SARS • Obtain nearest neighbor panels for specificity testing • Additional SARS strains • Other coronaviruses USAMRIID

  18. LTC David Kulesh Bonnie Loveless, MS Deanna Christensen, MS SPC Elizabeth Bode George Ludwig, PhD Tammy Clements Len Wasieloski, PhD Melanie Ulrich, PhD Philip Craw Alexandra Zalles-Ganley Susan Coyne COL Timothy Endy, MD Jason Paragas, PhD John Huggins, PhD Lisa Hensley, PhD Elizabeth Fritz, PhD Aura Garrison, MS LCDR James Lawler, MD Acknowledgements USAMRIID

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