2013 Archived Content
Quantitative Real-Time PCR
Applications for Molecular Diagnostics
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Tuesday, February 12
8:00 am Morning Coffee
8:25 Chairperson’s Remarks
8:30 Detection of Bacterial and Fungal Pathogens Using Novel Platform Molecular Diagnostics Target Technologies
Terry Smith, Ph.D., Professor and Vice President for Research, Natural Sciences, National University of Ireland Galway
Detection and identification of bacterial and fungal pathogens using quantitative real time PCR assays provide a rapid, sensitive and specific alternative to traditional infectious disease diagnostic test methods. The Molecular Diagnostics Research Group (MDRG) at NUI Galway, Ireland, have identified, exemplified and patented a suite of novel molecular targets for microorganism identification. Using the molecular targets identified, including the ssrA (RiboSEQ), rps 7 (MycoSEQ) and LepA (RiboTech) gene targets, a number of nucleic acid tests (NAT) for the sensitive and specific identification of bacterial and fungal pathogens of clinical significance have been developed. While numerous assays for organisms from a range of clinical fields have been developed, this presentation will outline real-time PCR –based assays for the organisms associated with hospital acquired and respiratory tract infections, including for example: Pseudomonas aeruginosa; Klebsiella pneumonia; Enterobacter aerogenes; Bordetella pertussis; Staphylococcus aureus; Candida species; and Aspergillus species.
9:00 Next-Generation PCR
Uffe Vest Schneider, M.D., Ph.D., CSO, QuantiBact A/S
TINA modified PCR primers improves efficacy and robustness of end-point as well as real-time PCR. The efficacy is improved in terms of stable PCR performance over a wide range of primer concentrations and annealing temperatures, shorter cycling time and improved sensitivity. The design rules for TINA modified primers are very simple, sequence independent, constant and reproducible. TINA modified primers are synthesized by conventional oligonucleotide synthesis. This presentation outlines when, why and how to use TINA modified primers in PCR.
9:30 Enhancing Genetic Analysis Through Droplet Digital PCRYann Jouvenot, Ph.D., Staff Scientist, Bio-Rad LaboratoriesDroplet digital PCR (ddPCR) is a method for absolute quantification of nucleic acid molecules with extreme precision and accuracy. With a sensitivity as low as 10%, the QX100 ddPCR system from Bio-Rad provides a revolutionary approach to DNA quantification. It is particularly useful for studies of copy number variations (CNV), rare mutation detection, single-cell studies and characterization of NGS libraries.
10:00 Coffee Break with Exhibit and Poster Viewing
10:30 Intraoperative PCR for Detection of Metastatic Head and Neck Cancer
Robert L. Ferris, M.D., Ph.D., FACS, Professor and Chief, Division of Head and Neck Surgery; Associate Director, Translational Research; Co-Leader, Cancer,Immunology Program, University of Pittsburgh Cancer Institute
This outlines the selection of relevant tumor gene markers, design and validation of their concordance using PCR with standard pathologic detection of metastatic carcinoma in regional lymph nodes, and the potential clinical application of PCR for nodal assessment within an intraoperative time frame.
11:00 Application of Taqman Array Cards (TAC) for Multipathogen Detection in Clinical Specimens
Jonas Winchell, Ph.D., Laboratory Chief, Pneumonia Response and Surveillance Laboratory, Division of Bacterial Diseases, Respiratory Diseases Branch, Centers for Disease Control and Prevention (CDC)
TAC is a microfluidic, RT-PCR multipathogen detection technology which the CDC used to rapidly screen for potential etiologies of unexplained respiratory disease outbreaks. CDC is spearheading a variety of studies designed to assess the utility of this technology in both domestic and international settings.
11:30 Droplet Digital PCR (ddPCR) as a Highly Sensitive Method for Copy Number Measurement in Cancer
Hatice Duzkale, Clinical Molecular Genetics Fellow, Harvard Medical School & Brigham and Women's Hospital
Laboratory for Molecular Medicine; Partners Healthcare Center for Personalized Genetic Medicine
We have validated ddPCR as a highly sensitive and accurate copy number estimation method in a CLIA-certified environment at Laboratory for Molecular Medicine. The data has been produced using DNA from formalin fixed paraffin embedded tissue of a variety of different cancer types, originating from cell lines or primary tumors, and compared with data acquired by FISH, MLPA and aCGH. Stepwise validation process, covering 12 target and 3 reference loci, and the data will be shared with the audience.
12:00 pm Close of Symposium
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