Development of standard method performance requirements for biological threat agent detection methods.

Standards and third-party testing are necessary to demonstrate the performance and limitations of biological threat agent (biothreat) detection technologies to allow appropriate response actions by end-users and responders. In order to address this need, the Department of Homeland Security, Science and Technology Directorate has funded AOAC INTERNATIONAL to develop standards and perform conformity assessment. AOAC formed the Stakeholder Panel on Agent Detection Assays to develop consensus performance criteria (standard method performance requirements; SMPRs) for methods that detect biothreats. This paper documents the development of the first five biothreat SMPRs, including the voluntary consensus process, the components of an SMPR, the use of SMPRs in developing validation protocols, and a description of the development efforts and considerations for each of the current SMPRs.

Comparison of the Modified Centers for Disease Control and Prevention 2019-Novel Coronavirus Real-Time RT-PCR Method for Detection of Infectious and Heat-Inactivated Virus on Stainless Steel.

BACKGROUND: Infectious Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) was used in the validation of methods for detection of SARS-CoV-2 on stainless-steel surfaces in the AOAC Research Institute Emergency Response Validation project. Handling infectious virus requires Biosafety Level (BSL)-3 facilities. OBJECTIVE: To compare the recovery and detection of infectious and heat-inactivated (HI; 65°C for 30 min) SARS-CoV-2 from stainless steel by the modified US Centers for Disease Control and Prevention (CDC) 2019-Novel Coronavirus (2019-nCoV) Real Time Reverse Transcription Polymerase Chain Reaction (RT-PCR) Diagnostic Panel. METHOD: Viral stocks were diluted in viral transport medium (VTM) and deposited onto stainless-steel test areas at 2 × 103 and 2 × 104 genomic copies for low and high, respectively. Test areas were sampled and aliquots of the resulting test solutions analyzed by RT-qPCR according to the CDC method. Results were analyzed by probability of detection (POD) statistics. RESULTS: The low level, where fractional positive results (25-75%) are expected, yielded PODI = 0.80 (0.58, 0.92) for the infectious virus and PODHI = 0.15 (0.05, 0.36) for the HI virus. The bias, dPODHI = -0.65 (-0.80, -0.35), demonstrated a statistical difference between infectious and HI virus detection. No difference was observed at the high inoculation level. CONCLUSIONS: Despite the statistical difference observed, the use of the HI virus is a viable alternative for matrix extension studies using a method comparison study design. Highlights: The use of HI SARS-CoV-2 can mitigate the need for a BSL-3 facility for matrix extension validation of alternative methods in SARS-CoV-2 studies. HIGHLIGHTS: The use of HI SARS-CoV-2 can mitigate the need for a BSL-3 facility for matrix extension validation of alternative methods in SARS-CoV-2 studies.

Performance Tested Method multiple laboratory validation study of ELISA-based assays for the detection of peanuts in food.

Performance Tested Method multiple laboratory validations for the detection of peanut protein in 4 different food matrixes were conducted under the auspices of the AOAC Research Institute. In this blind study, 3 commercially available ELISA test kits were validated: Neogen Veratox for Peanut, R-Biopharm RIDASCREEN FAST Peanut, and Tepnel BioKits for Peanut Assay. The food matrixes used were breakfast cereal, cookies, ice cream, and milk chocolate spiked at 0 and 5 ppm peanut. Analyses of the samples were conducted by laboratories representing industry and international and U.S governmental agencies. All 3 commercial test kits successfully identified spiked and peanut-free samples. The validation study required 60 analyses on test samples at the target level 5 microg peanut/g food and 60 analyses at a peanut-free level, which was designed to ensure that the lower 95% confidence limit for the sensitivity and specificity would not be <90%. The probability that a test sample contains an allergen given a prevalence rate of 5% and a positive test result using a single test kit analysis with 95% sensitivity and 95% specificity, which was demonstrated for these test kits, would be 50%. When 2 test kits are run simultaneously on all samples, the probability becomes 95%. It is therefore recommended that all field samples be analyzed with at least 2 of the validated kits.