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Quantitative Real-Time PCR for Molecular Diagnostics - Day 2

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FRIDAY, FEBRUARY 17           

8:00 am Breakfast Breakout Discussion Groups

In this interactive session, several topics will be offered for discussions and delegates are invited to choose a breakout topic of interest and join the moderated discussion at hand. In this informal setting, participants are encouraged to share examples from their work, vet ideas with peers and be part of a group problem-solving endeavor. We emphasize that this discussion is an informal exchange amongst scientists and is not meant to be, in any way, a product discussion.

Topic 1: Nucleic Acid Analysis by Digital PCR

Moderator: Feng Shen, Ph.D., Director, Research and Development, SlipChip LLC 

Topic 2: Finding Reference Genes for Normalization

Moderator: Virginia Rebecca Falkenberg, Ph.D., Centers for Disease Control and Prevention (CDC)

Topic 3: RT-PCR Assays for Infectious Diseases

Moderator: Edwin Kamau, Ph.D, Chief, Molecular Diagnostics, Malaria Vaccine Development, Walter Reed Army Institute of Research


9:10 Chairperson’s Opening Remarks 


Advances in Detection of Infectious Disease

Carl Wittwer, M.D., Professor, Pathology, University of Utah 

10:00 Assay for the Analysis of Total Nucleic Acids by qRT-PCR for Detection of Low Parasite Levels in Patients

Edwin Kamau, Ph.D, Chief, Molecular Diagnostics, Malaria Vaccine Development, Walter Reed Army Institute of Research

I will present our experience in validation of real-time PCR assays for detection of infectious diseases; dengue virus and malaria Plasmodium parasite.

10:30 Coffee Break in the Exhibit Hall with Poster Viewing

11:15 Rapid Diagnosis of Respiratory Viral Infections in Asthma Patients Using an Integrated Sample Prep and qPCR Technology

Preveen Ramamoorthy, Ph.D., Director, Molecular Diagnostics, Advanced Diagnostic Laboratories; Assistant Professor, Department of Medicine, National Jewish Health

My talk will focus on the clinical impact of respiratory viral infections in asthma patients and our experience in implementing an integrated sample prep and qPCR technology for rapid diagnosis of respiratory infection. The results of our clinical and analytical validation study using this 1 hour test that can diagnose 20 respiratory viruses will be presented.

11:45 The Challenge of Accurate Pathogen Quantitation

David R. Hillyard, M.D., Director, Molecular Infectious Disease Testing, ARUP Laboratories

12:15 pm Lunch on Your Own

1:55 Chairperson’s Remarks

2:00 Re-Engineering Microbial Genomes

Peter Carr, Ph.D., Senior Research Staff, Lincoln Laboratory, Massachusetts Institute of Technology

2:30 SASqPCR for Robust and Rapid Analysis of RT-qPCR

Daijun Ling, Ph.D., Scientist, USC Davis School of Gerontology, University of Southern California
RT-qPCR is widely used for quantification of gene expression in response to various biomedical conditions. Analysis of RT-qPCR data requires many iterative computations for data normalization and analytical optimization. This presentation will introduce a computer program based on statistical software SAS, SASqPCR, for robust and rapid RT-qPCR data analysis. SASqPCR can make easier for reference selection, data normaliztion and analysis.  I may primarily focus on how this program was developed, how to use and what advanage of this program has for RT-qPCR data analysis. The rationale for data normalization and reference gene selection for robust RT-qPCR quantification will also be included.

3:00 Refreshment Break in the Exhibit Hall with Poster Viewing

3:30 LNA-Eenhanced Real-Time ice-COLD-PCR for Ultra-Sensitive Detection of Low-Level Lung Cancer Resistance Mutations

G. Mike Makrigiorgos, Ph.D., Director, Biophysics Laboratory and Medical Physics Division, Dana Farber Cancer Institute, Harvard Medical School

Conventional real time PCR is limited in its ability to identify and quantify low-level mutant DNA alleles within a high excess of wild type DNA. Yet, detection of these rare mutations can be clinically highly significant (e.g. in the field of cancer). We present COLD-PCR and ice-COLD-PCR, technologies that boost the sensitivity of almost all diagnostic assays including conventional real time PCR by 100-fold when it comes to detecting low-level mutations. Examples for detecting EGFR and p53 mutations, that cause resistance to chemotherapy, will be presented.

4:00 Real-time PCR and high reSolution Melt Analysis for Rapid Detection of Mycobacterium leprae Drug Resistance Mutations and Strain Types

Wei Li Ph.D., Scientist, Leprosy Research Laboratories, Dept of Microbiology, Immunology,& Pathology, Colorado State University

Leprosy is still endemic in many countries. The objectives of our research are to characterize the clinical isolates of Mycobacterium leprae to understand the factors leading to to new cases and transmission of leprosy. We developed Real-Time PCR based high resolution melt to 1) estimate the genetic factors of the pathogen causing the primary drug resistance to dapson, rifampicin and fluoroquinolones, the components of multidrug (MDT) in the endemic regions 2) develop cost effective technologies to expedite global drug resistance surveillance (DRS) 3) to develop assays for detection and quantitation of M. leprae DNA in clinical specimens, and 4) identify and trace strain types of M. leprae in endemic populations.

4:30 Circulating Mitochondrial DNA as a Non-Invasive Biomarker for Mitochondrial Dysfunction: Accurate Quantification with our Co-Amplification of Nuclear Pseudogenes and Dilution Bias

Afshan Malik, Ph.D., Lecturer, Diabetes Research Group, Division of Diabetes and Nutrition, King’s College London

Changes in mitochondrial DNA (MtDNA) may be indicative of mitochondrial dysfunction, the latter central to numerous diseases of oxidative stress. Alterations in MtDNA content have been found in a broad range of human diseases, such as diabetes and its complications, obesity, cancer, HIV complications, ageing and others. However the methodology being used to measure MtDNA needs to be improved as >90% of the mitochondrial genome is duplicated in the nuclear genome leading to co-amplification of nuclear pseudogenes and therefore inaccurate quantification of MtDNA when qPCR is utilized. We have developed an accurate method which overcomes this and other problems with MtDNA qPCR.

5:00 End of Conference

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