Evaluation of rapid and sensitive DNA extraction methods for detection of cytomegalovirus in dried blood spots

Abstract

BackgroundDried blood spots (DBS), collected universally from newborns in the U.S., could be used as a matrix for the detection of cytomegalovirus (CMV) infection in infants. However, sensitivity to detect CMV in DBS as compared to saliva and urine is variable across studies largely due to the DNA extraction method. Thermal shock, a widely used DNA extraction method, is highly sensitive for the detection of CMV in DBS, however, the processing time required is not practical for high-throughput testing. ObjectiveTo determine if rapid and cost-effective DNA extraction methods amenable to newborn screening (NBS) could achieve the same sensitivity as the thermal shock method. Study designDBS were prepared from CMV positive blood samples from 20 organ transplant recipients. Three DNA extraction methods were compared for relative yield and sensitivity of detection of CMV DNA: thermal shock, KOH Tris buffer, and DNA Extract All. CMV DNA was detected by real-time quantitative polymerase chain reaction (qPCR). ResultsThe KOH Tris and DNA Extract All methods gave higher yields and sensitivity of CMV detection in DBS than thermal shock, which were significantly greater when viral loads were ≤ 10,000 copies/ml blood. Both methods gave faster turnaround times than thermal shock and would be better suited for NBS. ConclusionsThe choice of DNA extraction method greatly influences the ability to detect low levels of CMV DNA in DBS. Moreover, development of highly sensitive yet rapid methods for CMV detection could help facilitate future newborn screening of CMV in DBS.