Display Settings:

Format

Send to:

Choose Destination
We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
J Biomol Tech. 2011 Apr;22(1):5-9.

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

Author information

  • 1Women’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

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:

cytomegalovirus, dried blood spots, nPCR

PMID:
21455476
[PubMed - indexed for MEDLINE]
PMCID:
PMC3059536
Free PMC Article
PubMed Commons home

PubMed Commons

0 comments
How to join PubMed Commons

    Supplemental Content

    Full text links

    Icon for PubMed Central
    Loading ...
    Write to the Help Desk