Pre-PCR processing in bioterrorism preparedness: improved diagnostic capabilities for laboratory response networks
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Johannes Hedman1, Rickard Knutsson, Ricky Ansell
1Johannes Hedman, PhD, is employed as a specialist at the Swedish National Laboratory of Forensic Science (SKL) and holds a research position at Applied Microbiology, Lund University. Rickard Knutsson, PhD, is Director of Security Department, National Veterinary Institute (SVA), Uppsala, Sweden . Ricky Ansell, PhD, is employed as forensic advisor and senior reporting officer at the Swedish National Laboratory of Forensic Science (SKL). Birgitta Rasmusson, PhD, is employed as research director at the Swedish National Laboratory of Forensic Science (SKL) and holds a position as adjunct professor at Applied Microbiology, Lund University. Peter Rådström, PhD, is employed as professor Applied Microbiology, Lund University.
Abstract:
Diagnostic DNA analysis using polymerase chain reaction (PCR) has become a valuable tool for rapid detection of biothreat agents. However, analysis is often challenging because of the limited size, quality, and purity of the biological target. Pre-PCR processing is an integrated concept in which the issues of analytical limit of detection and simplicity for automation are addressed in all steps leading up to PCR amplification--that is, sampling, sample treatment, and the chemical composition of PCR. The sampling method should maximize target uptake and minimize uptake of extraneous substances that could impair the analysis--so-called PCR inhibitors. In sample treatment, there is a trade-off between yield and purity, as extensive purification leads to DNA loss. A cornerstone of pre-PCR processing is to apply DNA polymerase-buffer systems that are tolerant to specific sample impurities, thereby lowering the need for expensive purification steps and maximizing DNA recovery. Improved awareness among Laboratory Response Networks (LRNs) regarding pre-PCR processing is important, as ineffective sample processing leads to increased cost and possibly false-negative or ambiguous results, hindering the decision-making process in a bioterrorism crisis. This article covers the nature and mechanisms of PCR-inhibitory substances relevant for agroterrorism and bioterrorism preparedness, methods for quality control of PCR reactions, and applications of pre-PCR processing to optimize and simplify the analysis of various biothreat agents. Knowledge about pre-PCR processing will improve diagnostic capabilities of LRNs involved in the response to bioterrorism incidents.
