Cross-Institute Evaluations of Inhibitor-Resistant PCR Reagents for Direct Testing of Aerosol and Blood Samples Containing Biological Warfare Agent DNA

Abstract

The down-selected reagents underwent further testing. In the United Kingdom experiments, both reagents were tested against seven contrived aerosol collector samples containing B. anthracis Ames DNA and B. subtilis spores from a commercial formulation (BioBall). In PCR assays with reaction mixtures containing 40% crude sample, an airfield-collected sample induced inhibition of the B. subtilis PCR with the KAPA reagent and complete failure of both PCRs with the Fast Virus reagent. However, both reagents allowed successful PCR for all other samples-which inhibited PCRs with a non-inhibitor-resistant reagent. In the United States, a cross-assay limit-of-detection (LoD) study in blood was conducted. The KAPA Blood Direct reagent allowed the detection of agent DNA (by four PCRs) at higher concentrations of blood in the reaction mixture (2.5%) than the Fast Virus reagent (0.5%), although LoDs differed between assays and reagent combinations. Across both groups, the KAPA Blood Direct reagent was determined to be the optimal reagent for inhibition relief in PCR.

Comparative analysis of the sensitivity of metagenomic sequencing and PCR to detect a biowarfare simulant (Bacillus atrophaeus) in soil samples

“…These experiments also demonstrated that the PCR DNA yield decreased when the amount of extract was higher than 2.4 μl. In line with previous interpretation for experiments carried out on environmental samples [24–27], we concluded that the DNA extracts contained PCR inhibitors and we used limited amounts of the DNA extracts in subsequent experiments.…”

Evaluation of the Biofire FilmArray BioThreat-E Test (v2.5) for Rapid Identification of Ebola Virus Disease in Heat-Treated Blood Samples Obtained in Sierra Leone and the United Kingdom

“…Although FilmArray control PCRs performed on the discrepant sample were successful, the performance of these assays is measured by the system using only qualitative melt-curve data (5); therefore, an increase in the control PCR C q value, which might indicate a general inhibition effect and therefore explain the EBOV negative, would not be detected by the system. Individual PCRs are known to be differentially affected by the same inhibitor (16), supporting the hypothesis of a control PCR success but EBOV PCR failure.…”

Current Trends in the Biosensors for Biological Warfare Agents Assay

“…The PCR is dependent on used primers, which are responsible for the specificity of the method. Various configurations of the method are available from which real time PCR is likely the most widely used in the current praxis [16,17]. There are also commercially available PCR devices suitable for field assay.…”