Quantitative Real-Time PCR
Applications for Molecular Diagnostics
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Quantitative real-time PCR is a valuable technology used for diagnostics of diseases such as cancer and infectious diseases, and for the detection of bacterial, fungal and viral pathogens. The introduction of novel technology platforms such as digital PCR, high-throughput platforms and improvements in automation and standardization may provide useful tools for diagnosis, prognosis and therapeutic evaluations for both the pharmaceutical industry and the medical community to move the application of qPCR to the next level. Issues such as reference genes and their reliability for quantitative results will be addressed. This meeting is designed to present the advancements and recent progress of qPCR in diagnostics, and case studies to highlight the applications of the technology.
Monday, February 11
7:30 am Registration and Morning Coffee
8:25 Chairperson’s Opening Remarks
8:30 Anchor Primers for the Detection of Extremely Rare Sequence Variants
Fred Russell Kramer, Ph.D., Professor, Department of Microbiology and Molecular Genetics, Public Health Research Institute, New Jersey Medical School
9:00 Sensitive Platform for FUS-CHOP Transcripts Detection in Human Liposarcoma
Nitin S. Patil, Ph.D., Scientist, Molecular Oncology of Solid Tumors -Joint Unit, DKFZ (German Cancer Research Center) Heidelberg
We describe a method using RT-PCR to identify and differentiate the fusion transcripts formed in the t(12;16)(q13;p11) chromosomal translocation. This was achieved using transcript individualized primers/probes, designed to detect specifically different variants in both frozen and FFPE tissues.
9:30 Quantification of RNA Degradation and its Use for Correct Quantification of RNA Transcript Number
Alexander Morley, M.D., Professor, Head, Minimal Residual Disease Group, Haematology & Genetic Pathology, Flinders University
Wedeveloped a simple PCR-based method for quantification of degradation and using the result to obtain the true level of mRNA transcripts in a sample. Compared to electrophoretic methods this is quantitative, more sensitive, quicker, cheaper and informative over a broader range of degradation.
10:00 Coffee Break with Exhibit and Poster Viewing
10:30 Technical Considerations for Diagnostic RT-QPCR
Chaminda Selgado, Ph.D., Head of Department, CMC Bioassay & Genomics, NDA-Analytics
This is a high level overview of the stages and issues associated with developing a RT-QPCR based diagnostic assay/ platform. The areas will cover sample storage, nucleic acid purification, reverse transcription strategies, real-time chemistry, platform choice, consumables, and analysis considerations.
11:00 Harmonized Quantification of the BCR-ABL Transcripts using Certified Reference Materials
Philippe Corbisier, Ph.D., Scientific Project Manager, Reference Material Unit, European Commission
For the first time DNA material has been certified for its absolute copy number at levels of 10 copies /µL. This material will allow to harmonize the quantification of BCR-ABL transcripts and allow a reliable diagnostic of chronic myeloid leukemia.
11:30 Democratization of Molecular Diagnostics: Bringing Simplified Multiplex Real Time PCR Assays to a Hospital Near You
Scott C. Johnson, Ph.D., Vice President, Product Development & Manufacturing, Luminex CorporationThe latest developments of Luminex’s upcoming open-platform sample-to-answer molecular diagnostic system will be discussed, including how a clear focus on end-user needs will enable this technology to bring Molecular Dx capabilities to more hospitals and labs than before.
12:00 pm Luncheon Presentation: Absolute DNA Copy Number Without StandardsJohn SantaLucia, Ph.D., CEO & President, DNA Software, Inc.qPCR CopyCount is a new cloud-based product that analyzes the shape of a qPCR curve to reveal the absolute copy number of DNA at cycle zero without the use of a standard curve. This uses a new principle called “counting PCR” in which each copy of DNA is literally counted at each cycle of PCR.
1:25 Chairperson’s Remarks
1:30 Highly Divergent Gene Families are Efficiently Amplified Using Low-Concentration Initiator Primers
Kenneth E. Pierce, Ph.D., Senior Research Scientist, Department of Biology, Brandeis University
Detecting sequences of divergent bacterial or viral genes can be challenging. We demonstrate consistent amplification of each subfamily of the CTX-M antibiotic resistance genes using low concentration initiator primers (i Primers) in combination with a single pair of consensus LATE-PCR primers.
2:00 TOCE: A Technology for Changing the Molecular Diagnostics Paradigm
David L. Dolinger, Ph.D., Executive Vice President, Business Development & Technology Realization, Seegene, Inc.
A new chemistry has been developed to fully exploit real-time PCR in high multiplex analysis. This chemistry provides the ability to detect multiple targets in a single fluorescence channel. TOCE is a highly specific and versatile chemistry that greatly expands the capabilities of currently installed widely-used real-time platforms.
2:30 New Developments in Detection of Rare Mutations Using Real Time COLD-PCR and DiSSeCT Technology
G. Mike Makrigiorgos, Ph.D., Director, Biophysics Laboratory and Medical Physics Division, Dana Farber Cancer Institute, Harvard Medical School
Multiplex detection of low-level mutant alleles in the presence of wild-type DNA is very useful e.g. for cancer, prenatal diagnosis and infectious diseases. We present new technologies, DiSSeCT and COLD-PCR that, when combined lead to detection of traces of mutations in cancer and circulating DNA.
3:00 Refreshment Break with Exhibit and Poster Viewing
3:30 Sloppy Molecular Beacon®: A New Paradigm in Rapid Pathogen Identification and Molecular Drug Susceptibility Testing
Soumitesh Chakravorty, Ph.D., Instructor, Center for Emerging Pathogens, NJMS, UMDNJ
Sloppy molecular beacons are mis-match tolerant fluorescent DNA probes which can identify a wide range of DNA sequences by generating probe-target hybrid “Tm signatures”. This approach enables rapid and definitive identification of bacterial pathogens and MDR and XDR tuberculosis.
4:00 Considerations for Using Digital PCR as a Diagnostics Tool
Alexandra S. Whale, Ph.D., Researcher, LGC Ltd.
dPCR offers the potential to revolutionize molecular diagnostics by improving technical reproducibility and analytical sensitivity, all using an unambiguous digital output. We are defining dPCR efficacy to provide guidelines for using this approach in order to facilitate maximum diagnostic impact.
4:30 Digital PCR Capabilities vs. Cost
Reginald Beer, Ph.D., Medical Diagnostics Initiative Leader, Center for Micro and Nanotechnologies, Lawrence Livermore National Laboratories
Digital PCR has shown promise in tumor and metastasis detection, copy number variation, and absolute quantitation, but it is still widely misunderstood, expensive, and dependent on the prerequisite amplification. In this talk we discuss costs and benefits of digital PCR.
5:00 Breakout Discussions:
Certified Reference Materials in QPCR
Moderator: Philippe Corbisier, Ph.D., Scientific Project Manager, Reference Material Unit, European Commission
Are you using any reference material? if yes which type ?
Are you confronted to provided traceable results?
Harmonisation or validation of procedure with the help of a reference material – how common is it?
What level of precision is required for a particular application?
PCR in Medical Diagnostics
Moderator: Reginald Beer, Ph.D., Medical Diagnostics Initiative Leader, Center for Micro and Nanotechnologies, Lawrence Livermore National Laboratories
What titer accuracy is required (not just desired) in medical applications?
How would you prioritize speed, titer accuracy, sensitivity, specificity in the clinic? In the ER and ICU?
What degree of multiplexing is required?
6:00 Close of Day
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