Please use this identifier to cite or link to this item:
|Title:||Comparison of DNA extraction methods from small samples of newborn screening cards suitable for retrospective perinatal viral research|
|Citation:||Journal of Biomolecular Techniques, 2011; 22(1):5-9|
|Publisher:||The Association of Biomolecular Resource Facilities|
|Organisation:||South Australian Cerebral Palsy Research Group|
|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|
|Appears in Collections:||Obstetrics and Gynaecology publications|
Cerebral Palsy Research Group publications
Files in This Item:
There are no files associated with this item.
Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.