Evaluation of DNA extraction methods for the detection of Cytomegalovirus in dried blood spots

Abstract

Background Dried blood spots (DBS) are collected universally from newborns and may be valuable for the diagnosis of congenital Cytomegalovirus (CMV) infection. The reported analytical sensitivity for DBS testing compared to urine or saliva varies greatly across CMV studies. The purpose of this study was to directly compare the performance of various DNA extraction methods for identification of CMV in DBS including those used most often in CMV studies. Study design Whatman® Grade 903 filter paper cards were spotted with blood samples from 25 organ transplant recipients who had confirmed CMV viremia. Six DNA extraction methods were compared for relative yield of viral and cellular DNA: 2 manual solution-based methods (Gentra Puregene, thermal shock), 2 manual silica column-based methods (QIAamp DNA Mini, QIAamp DNA Investigator), and 2 automated methods (M48 MagAttract Mini, QIAcube Investigator). DBS extractions were performed in triplicate followed by real-time quantitative PCR (qPCR). Results For extraction of both viral and cellular DNA, two methods (QIAamp DNA Investigator and thermal shock) consistently gave the highest yields, and two methods (M48 MagAttract Mini and QIAamp DNA Mini) consistently gave the lowest yields. There was an average 3-fold difference in DNA yield between the highest and lowest yield methods. Conclusion The choice of DNA extraction method is a major factor in the ability to detect low levels of CMV in DBS and can largely account for the wide range of DBS sensitivities reported in studies to date.

Viral Loads in Congenital Cytomegalovirus Infection From a Highly Immune Population

“…Different specimens were collected on the same day from an infant if enrolled. DNA was eluted from swabs with Extracta (Quanta, United States) and extracted from a 6 mm-diameter DBS punches using thermal shock [4, 5]. Detection of CMV DNA was performed with real-time PCR with TaqMan-based primers and probes targeting the glycoprotein B gene on Mx3000P qPCR Systems [4, 5].…”

“…DNA was eluted from swabs with Extracta (Quanta, United States) and extracted from a 6 mm-diameter DBS punches using thermal shock [4, 5]. Detection of CMV DNA was performed with real-time PCR with TaqMan-based primers and probes targeting the glycoprotein B gene on Mx3000P qPCR Systems [4, 5]. The commercially standardized human CMV DNA quantitated with digital PCR by Advanced Biotechnologies, Inc. (Eldersburg, MD, USA) was included on every PCR plate to quantify the viral loads.…”

Acquired Hearing Loss in Children

“…20 Positive PCR using dried blood spots can confirm a diagnosis of congenital CMV outside the newborn period when viral culture or CMV IgG cannot differentiate between congenital and postnatally acquired infection. [21][22][23][24][25] In addition, PCR technology may become a basis for large-scale screening.…”

Evaluation of the Automated QIAsymphony SP/AS Workflow for Cytomegalovirus DNA Extraction and Amplification from Dried Blood Spots

“…Studies reporting detection of CMV from DBS have already been performed [6,7,12,16,17,18,19]. However, methods reporting the highest sensitivities for CMV DNA detection from DBS are not suitable for high-throughput screening being labor intensive and not cost effective.…”