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

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.