RAZOR EX Anthrax Air Detection System for detection of Bacillus anthracis spores from aerosol collection samples: collaborative study.

The RAZOR EX Anthrax Air Detection System was validated in a collaborative study for the detection of Bacillus anthracis in aerosol collection buffer. Phosphate-buffered saline was charged with 1 mg/mL standardized dust to simulate an authentic aerosol collection sample. The dust-charged buffer was spiked with either B. anthracis Ames at 2000 spores/mL or Bacillus cereus at 20 000 spores/mL. Twelve collaborators participated in the study, with four collaborators at each of three sites. Each collaborator tested 12 replicates of B. anthracis in dust-charged buffer and 12 replicates of B. cereus in dust-charged buffer. All samples sets were randomized and blind-coded. All collaborators produced valid data sets (no collaborators displayed systematic errors) and there was only one invalid data point. After unblinding, the analysis revealed a cross-collaborator probability of detection (CPOD) of 1.00 (144 positive results from 144 replicates, 95% confidence interval 0.975-1.00) for the B. anthracis samples and a CPOD of 0.00 (0 positive results from 143 replicates, 95% confidence interval 0.00-0.0262) for the B. cereus samples. These data meet the requirements of AOAC Standard Method Performance Requirement 2010.003, developed by the Stakeholder Panel on Agent Detection Assays.

RAZOR EX anthrax air detection system.

The RAZOR EX Anthrax Air Detection System, developed by Idaho Technology, Inc. (ITI), is a qualitative method for the detection of Bacillus anthracis spores collected by air collection devices. This system comprises a DNA extraction kit, a freeze-dried PCR reagent pouch, and the RAZOR EX real-time PCR instrument. Each pouch contains three assays, which distinguish potentially virulent B. anthracis from avirulent B. anthracis and other Bacillus species. These assays target the pXO1 and pXO2 plasmids and chromosomal DNA. When all targets are detected, the instrument makes an "anthrax detected" call, meaning that virulence genes of the anthrax bacillus are present. This report describes results from AOAC Method Developer (MD) and Independent Laboratory Validation (ILV) studies, which include matrix, inclusivity/exclusivity, environmental interference, upper and lower LOD of DNA, robustness, product consistency and stability, and instrument variation testing. In the MD studies, the system met the acceptance criteria for sensitivity and specificity, and the performance was consistent, stable, and robust for all components of the system. For the matrix study, the acceptance criteria of 95/96 expected calls was met for three of four matrixes, clean dry filters being the exception. Ninety-four of the 96 clean dry filter samples tested gave the expected calls. The nucleic acid limit of detection was 5-fold lower than AOAC's acceptable minimum detection limit. The system demonstrated no tendency for false positives when tested with Bacillus cereus. Environmental substances did not inhibit accurate detection of B. anthracis. The ILV studies yielded similar results for the matrix and inclusivity/exclusivity studies. The ILV environmental interference study included environmental substances and environmental organisms. Subsoil at a high concentration was found to negatively interfere with the pXO1 reaction. No interference was observed from the environmental organisms. The nucleic acid LOD, however, was 10 times higher (1 pg/reaction, equivalent to about 200 spores) than that found in the MD study. These results indicate that the RAZOR System is a sensitive and specific system that accurately identifies B. anthracis in aerosol matrixes and in the presence of interfering substances, and that the method can be performed by an independent laboratory and achieve similar results.