title: Comparison of DNA extraction methods from small samples of newborn screening cards suitable for retrospective perinatal viral research.
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.
authors: Women’s and Children’s Hospital, Discipline of Obstetrics and Gynaecology, 1st Floor, Queen Victoria Bldg., 72 King William Rd., North Adelaide, SA 5006, Australia. gai.mcmichael@adelaide.edu.au
authors: Gai L
authors: GL
authors: McMichael
authors: Amanda R
authors: AR
authors: Highet
authors: Catherine S
authors: CS
authors: Gibson
authors: Paul N
authors: PN
authors: Goldwater
authors: Michael E
authors: ME
authors: O'Callaghan
authors: Emily R
authors: ER
authors: Alvino
authors: Alastair H
authors: AH
authors: MacLennan
mesh_terms: D000704:Analysis of Variance; D053000:Analytic Sample Preparation Methods; D001800:Blood Specimen Collection / Q000379:methods; D003587:Cytomegalovirus / Q000302:isolation & purification*; D003586:Cytomegalovirus Infections / Q000097:blood / Q000175:diagnosis; D004279:DNA, Viral / Q000097:blood* / Q000302:isolation & purification*; D005260:Female; D004854:Herpesvirus 4, Human / Q000302:isolation & purification; D006801:Humans; D007231:Infant, Newborn; D015997:Neonatal Screening / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011247:Pregnancy; D011251:Pregnancy Complications, Infectious / Q000821:virology; D012189:Retrospective Studies; D012680:Sensitivity and Specificity; D019562:Viral Load
chemical_list: D004279:DNA, Viral