Comparison of DNA extraction methods from small samples of newborn screening cards suitable for retrospective perinatal viral research

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dc.contributor.authorMcMichael, G.
dc.contributor.authorHighet, A.
dc.contributor.authorGibson, C.
dc.contributor.authorGoldwater, P.
dc.contributor.authorO'Callaghan, M.
dc.contributor.authorAlvino, E.
dc.contributor.authorMacLennan, A.
dc.contributor.organisationSouth Australian Cerebral Palsy Research Group
dc.date.issued2011
dc.description.abstractReliable detection of viral DNA in stored newborn screening cards (NSC) would give important insight into possible silent infection during pregnancy and around birth. We sought a DNA extraction method with sufficient sensitivity to detect low copy numbers of viral DNA from small punch samples of NSC. Blank NSC were spotted with seronegative EDTA-blood and seropositive EBV EDTA-blood. DNA was extracted with commercial and noncommercial DNA extraction methods and quantified on a spectrofluorometer using a PicoGreen dsDNA quantification kit. Serial dilutions of purified viral DNA controls determined the sensitivity of the amplification protocol, and seropositive EBV EDTA-blood amplified by nested PCR (nPCR) validated the DNA extraction methods. There were considerable differences between the commercial and noncommercial DNA extraction methods (P=0.014; P=0.016). Commercial kits compared favorably, but the QIamp DNA micro kit with an added forensic filter step was marginally more sensitive. The mean DNA yield from this method was 3 ng/μl. The limit of detection was 10 viral genome copies in a 50-μl reaction. EBV nPCR detection in neat and 1:10 diluted DNA extracts could be replicated reliably. We conclude that the QIamp Micro DNA extraction method with the added forensic spin-filter step was suitable for retrospective DNA viral assays from NSC.
dc.description.statementofresponsibilityGai L. McMichael, Amanda R. Highet, Catherine S. Gibson, Paul N. Goldwater, Michael E. O’Callaghan, Emily R. Alvino and Alastair H. MacLennan for the South Australian Cerebral Palsy Research Group
dc.identifier.citationJournal of Biomolecular Techniques, 2011; 22(1):5-9
dc.identifier.issn1524-0215
dc.identifier.issn1943-4731
dc.identifier.orcidMcMichael, G. [0000-0002-6811-5301]
dc.identifier.orcidGoldwater, P. [0000-0003-4822-8488]
dc.identifier.orcidO'Callaghan, M. [0000-0001-5038-5859] [0000-0002-8178-9714]
dc.identifier.urihttp://hdl.handle.net/2440/63738
dc.language.isoen
dc.publisherThe Association of Biomolecular Resource Facilities
dc.rightsCopyright © 2011 Association of Biomolecular Resource Facilities
dc.source.urihttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC3059536/?report=abstract&tool=pmcentrez
dc.subjectSouth Australian Cerebral Palsy Research Group
dc.subjectHumans
dc.subjectCytomegalovirus
dc.subjectHerpesvirus 4, Human
dc.subjectPregnancy Complications, Infectious
dc.subjectCytomegalovirus Infections
dc.subjectDNA, Viral
dc.subjectNeonatal Screening
dc.subjectBlood Specimen Collection
dc.subjectViral Load
dc.subjectAnalysis of Variance
dc.subjectSensitivity and Specificity
dc.subjectRetrospective Studies
dc.subjectPolymerase Chain Reaction
dc.subjectPregnancy
dc.subjectInfant, Newborn
dc.subjectFemale
dc.subjectAnalytic Sample Preparation Methods
dc.titleComparison of DNA extraction methods from small samples of newborn screening cards suitable for retrospective perinatal viral research
dc.typeJournal article
pubs.publication-statusPublished

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