Accession Number:

ADA461528

Title:

Evaluation of a Solid Phase DNA Binding Matrix for Downstream PCR Analysis

Descriptive Note:

Technical memorandum

Corporate Author:

DEFENCE RESEARCH AND DEVELOPMENT SUFFIELD (ALBERTA)

Report Date:

2005-12-01

Pagination or Media Count:

26.0

Abstract:

A commercially available solid-phase DNA binding matrix FTA cards was evaluated for its ability to capture and release DNA for downstream gene amplification and detection assays using polymerase chain reaction PCR analysis, as part of a project to determine the utility of FTA cards for sampling, archiving, and transport of samples that may contain biowarfare BW or bioterrorist BT agents. PCR was performed using assays designed to amplify and detect two different Bacillus anthracis virulence genes, namely the lethal toxin gene lef found on plasmid pXO1, and the capsule B gene capB, located on plasmid pXO2. PCR assays were conducted using real-time, Taqman fluorescent probe detection on a Cepheid Smart Cycler instrument. A baseline detection limit of about 300 gene copies was observed for each assay prior to DNA-FTA card binding studies using B. anthracis Ames DNA. Direct PCR of DNA bound to FTA discs resulted in a loss of sensitivity compared to DNA in solution however, this method proved better than the heat-elution method since PCR signals were observed at earlier cycles. Furthermore, FTA bound DNA generated a reproducible PCR signal for 12 of 12 replicate tests 100, compared to 11 of 12 92 for a single heat elution treatment, and 5 of 12 42 replicates for a double heat elution treatment.

Subject Categories:

  • Biochemistry
  • Chemical, Biological and Radiological Warfare

Distribution Statement:

APPROVED FOR PUBLIC RELEASE