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9/26/2014  |   4:30 PM - 6:00 PM   |  

Factors that Influence Sensitivity of Detection of Cytomegalovirus in Dried Blood Spots

Dried blood spots (DBS), which are collected universally from newborns, may be valuable in the diagnosis of congenital cytomegalovirus (CMV) infection in newborns with increased risk for hearing loss and cognitive disability. However, the reported sensitivity for DBS testing varies widely across CMV studies. Methods need to have sufficient sensitivity to identify infants who are asymptomatic at birth but at risk for sequelae. Our objective was to directly compare methods for extraction of CMV DNA from DBS and to optimize sensitivity for detection of CMV DNA. CMV DNA-positive whole blood from organ transplant recipients (n=25) with varying DNA viral loads was spotted onto Whatman® Grade 903 filter paper cards. Six DBS extraction methods were evaluated: Gentra Puregene, thermal shock, QIAamp DNA Mini kit, QIAamp DNA Investigator kit, and M48 Qiagen MagAttract. Recovery of CMV DNA was assessed by real-time quantitative PCR (qPCR). The yield of recovered DNA for each extraction method was used to assign specimens into low, intermediate or high viral load categories. We evaluated TaqMan® Universal Master Mix II and PerfeCTa® qPCR FastMix® II for effects on real-time qPCR sensitivity. In addition, twelve published primer and probe sets, which target different regions in the CMV genome were used to amplify serially diluted purified strain AD169 DNA to compare performance and limit of detection. Overall results showed that the QIAamp DNA Investigator kit and thermal shock extraction methods produced the highest yield of DNA and sensitivity of detection of CMV in DBS. We found that using these two methods in combination with qPCR reactions that contain PerfeCTa® qPCR FastMix® II and primer/probe sets with 100% homology to the target region gave the greatest sensitivity for CMV detection in DBS.

Deborah Koontz (Primary Presenter), duk5@cdc.gov;
Deborah Koontz, Ph.D., is a member of CDC’s Molecular Quality Improvement Program. She is the lead for the Cytogenetics and FISH laboratory and for molecular methods development for 22q11.2 deletion diagnosis and cytomegalovirus detection in dried blood spots. In the past, she has led large-scale population studies that involved medium to high-throughput genotyping of samples from various DNA collections to study diseases of public health significance. She has extensive experience in assay design and development of challenging genetic targets. Dr. Koontz earned her doctorate in Biology at Georgia Tech and was the manager of a molecular medicine laboratory specializing in the diagnosis of mitochondrial disorders and other neuromuscular diseases prior to joining CDC


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