Two multiplex PCR screening capabilities (TaqMan Array Cards and FilmArray) were evaluated for their ability to detect Bacillus anthracis, Francisella tularensis and Yersinia pestis in blood samples obtained from respective murine infection models. Blood samples were obtained from infected mice at 24 h intervals after exposure. Multiplex PCR results were compared with standard blood culture and singleplex real-time PCR. Across all three models, 71 mice were tested in total, within which a subset of 43 samples was shown to contain an infecting agent by at least one of the detection technologies. Within this subset of positive samples, for each model studied, the detection rates of each technology were compared. The B. anthracis model blood culture (14 of 15 agent-containing samples tested) and FilmArray PCR (12 of 15) were shown to have equivalent detection rates, which were significantly higher (at the 95 % confidence level) than singleplex (five of 14) or Array Card (two of 14) PCRs. The F. tularensis model blood culture (12 of 12) was shown to have a significantly higher (at 95 % confidence level) detection rate than all PCR technologies, with FilmArray (seven of 11) and singleplex (seven of 12) PCRs shown to have significantly higher (at 95 % confidence level) detection rates than the Array Card PCR (two of 11). Within the Y. pestis model, there was no significant difference in detection rates between blood culture (10 of 16), singleplex PCR (14 of 16), Array Card PCR (10 of 16) and FilmArray PCR (10 of 13).

Eight DNA extraction products or methods (Applied Biosystems PrepFiler Forensic DNA Extraction Kit; Bio-Rad Instagene Only, Bio-Rad Instagene & Spin Column Purification; EpiCentre MasterPure DNA & RNA Kit; FujiFilm QuickGene Mini80; Idaho Technologies 1-2-3 Q-Flow Kit; MoBio UltraClean Microbial DNA Isolation Kit; Sigma Extract-N-Amp Plant and Seed Kit) were adapted to facilitate extraction of DNA under BSL3 containment conditions. DNA was extracted from 12 common interferents or sample types, spiked with spores of Bacillus atropheaus. Resulting extracts were tested by real-time PCR. No one method was the best, in terms of DNA extraction, across all sample types. Statistical analysis indicated that the PrepFiler method was the best method from six dry powders (baking, biological washing, milk, plain flour, filler and talcum) and one solid (Underarm deodorant), the UltraClean method was the best from four liquids (aftershave, cola, nutrient broth, vinegar), and the MasterPure method was the best from the swab sample type. The best overall method, in terms of DNA extraction, across all sample types evaluated was the UltraClean method.

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