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Type: Journal article
Title: Comparison of DNA extraction methods from small samples of newborn screening cards suitable for retrospective perinatal viral research
Author: McMichael, G.
Highet, A.
Gibson, C.
Goldwater, P.
O'Callaghan, M.
Alvino, E.
MacLennan, A.
Citation: Journal of Biomolecular Techniques, 2011; 22(1):5-9
Publisher: The Association of Biomolecular Resource Facilities
Issue Date: 2011
ISSN: 1524-0215
Organisation: South Australian Cerebral Palsy Research Group
Statement of
Gai 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
Abstract: Reliable 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.
Keywords: South Australian Cerebral Palsy Research Group; Humans; Cytomegalovirus; Herpesvirus 4, Human; Pregnancy Complications, Infectious; Cytomegalovirus Infections; DNA, Viral; Neonatal Screening; Blood Specimen Collection; Viral Load; Analysis of Variance; Sensitivity and Specificity; Retrospective Studies; Polymerase Chain Reaction; Pregnancy; Infant, Newborn; Female; Analytic Sample Preparation Methods
Rights: Copyright © 2011 Association of Biomolecular Resource Facilities
RMID: 0020111713
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Appears in Collections:Obstetrics and Gynaecology publications
Cerebral Palsy Research Group publications

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