Real time PCR assay for the detection of CMV using eclipse hybridization probes

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Publication Type Poster
School or College School of Medicine
Department Pathology
Program ARUP Institute for Clinical and Experimental Pathology
Creator Hymas, Weston C.; Hillyard, David R.
Other Author Stevenson, Jeff
Title Real time PCR assay for the detection of CMV using eclipse hybridization probes
Date 2004-08-11
Description We developed a qualitative real time PCR assay to detect CMV in patient samples, using a novel hybridization probe termed the Eclipse probe (Epoch Biosciences). These are single probes with a fluorescent molecule attached to the 3' end and a dark quencher on the 5' end. Also conjugated to the 5' end of the probe is a minor groove binding moiety that stabilizes probe binding and allows much shorter probe sequences to be used. The assay is run on the HT7900 Sequence Detection System (Applied Biosystems) in a 96-well plate. We incorporated a heterologous internal control in the assay to monitor the extraction process and detect PCR inhibition. The internal control target is a plasmid, containing a fusion of GFP and C. elegans DNA. We validated ten sample types (spinal fluid, plasma, serum, amniotic fluid, ocular fluid, buffy coat, tissue, urine, bone marrow and bronchioalveolar lavage), and demonstrated the dependence that the limit of detection has on sample type. Since this assay went on-line last year, we have identified a number of patient samples that generated discrepant results between our qualitative test and the Roche CMV Amplicor Quantitative assay. Here we present the validation data for our real time assay, and results of the discrepant analysis.
Type Text
Publisher University of Utah
Subject Tissues Analysis; Elegans
Subject MESH Clinical Laboratory Techniques; Laboratory Techniques and Procedures; Blood Gas Analysis
Language eng
Bibliographic Citation Stevenson J, Hymas W.C. Hillyard D.R. A Real Time PCR Assay for the Detection of CMV Using Eclipse Hybridization Probes.
Format Medium application/pdf
Identifier ir-main,502
ARK ark:/87278/s683495h
Setname ir_uspace
Date Created 2012-06-13
Date Modified 2021-05-06
ID 702899
Reference URL
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