Evaluation of two multiplex real-time PCR screening capabilities for the detection of Bacillus anthracis, Francisella tularensis and Yersinia pestis in blood samples generated from murine infection models

Abstract

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).

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

“…One hundred femtograms of B. anthracis Ames strain DNA corresponds to 17.5 genome equivalents (3), and in a 20-l PCR mixture, 2.5% equates to a volume of 0.5 l. Extrapolating upward, 20 GEs in a 0.5-l volume would equate to a detectable agent concentration of 3.5 ϫ 10 4 CFU·ml Ϫ1 (17.5 ϫ 2,000) in whole blood. Although this is an improvement over the 10 6 -CFU·ml Ϫ1 level observed in a previous inhibitor resistance study with a Francisella tularensis PCR (17), an alternate study has demonstrated a pointof-care (PoC) PCR system, with integrated sample processing and DNA extraction, that is able to detect B. anthracis at sensitivities of 10 3 CFU·ml Ϫ1 and below in whole blood (29). Similarly, sample processing evaluations using the same PCR assays as those used here showed a relative sensitivity of 5 ϫ 10 1 CFU·ml Ϫ1 in whole blood when the nucleic acid was extracted (13).…”

Current and Emerging Legionella Diagnostics for Laboratory and Outbreak Investigations

“…TACs offer increased real-time PCR throughput (384 individual reactions) in a rapid, reproducible, and simple setup containing prespotted primer-and-probe combinations. This format has since been customized for larger field evaluations and for the detection of nonrespiratory syndromes (420)(421)(422)(423)(424). Mass spectrometry (MS) is a mature yet still evolving technology adapted to the rapid identification and classification of clinically relevant pathogens (for a review, see reference 425).…”

Influence of the length of target DNA overhang proximal to the array surface on discrimination of single-base mismatches on a 25-mer oligonucleotide array

“…Since this is an open platform, the user has control over the number of targets and the specific properties of the probes being used. The technology can also be used in a strip format for the simultaneous processing of up to 96 arrays, potentially allowing the development of higher throughput screening capabilities than other multiplex PCR-based platforms [23]. …”