A modified Heat-Shock Method for HCMV DNA Detection in Dried Blood Spots (DBS), Is It Efficient?

Congenital cytomegalovirus (CMV) infection is a major health issue not just because of symptomatic infection, but also due to late sequelae especially sensorineural hearing loss. Early detection of infection is critical for preventing and limiting disease in the absence of a licensed vaccination programme. Detection of CMV DNA in dried blood spots (DBS) routinely collected from newborns for metabolic disease screening has been suggested as a suitable method for universal CMV screening. The aim of this project is to investigate and explore methods of DNA extraction and amplification from DBS. Whole virus (CMV laboratory strain AD169) was used to spike a pathogen free EDTA human blood. DBS were prepared from this spiked blood. Two extraction methods were evaluated; a Qiagen column-based method (protocol 1) and a heat shock-based method (protocol 2). Following extraction, two different polymerase chain reaction (PCR) assays were used for DNA amplification. The sensitivity achieved using each extraction method and PCR assay was compared using serial tenfold dilutions of CMV DNA to determine the approximate quantitative cut-off values. Protocol 1 showed a 100 fold drop in sensitivity after spotting on DBS while protocol 2 could recover almost all DNA spotted on the DBS. The sensitivity of both CMV PCR assays was relatively high. In conclusion protocol 2 is a strong candidate for use during CMV universal screening, permitting detection of virus within the range of asymptomatic neonate’s peripheral blood viral load.