Multi-laboratory validation study of a real-time PCR method for detection of Salmonella in baby spinach.

The FDA Bacteriological Analytical Manual (BAM) Salmonella culture method takes at least 3 days for a presumptive positive result. The FDA developed a quantitative PCR (qPCR) method to detect Salmonella from 24-h preenriched cultures, using ABI 7500 PCR system. The qPCR method has been evaluated as a rapid screening method for a broad range of foods by single laboratory validation (SLV) studies. The present multi-laboratory validation (MLV) study was aimed to measure the reproducibility of this qPCR method and compare its performance with the culture method. Sixteen laboratories participated in two rounds of MLV study to analyze twenty-four blind-coded baby spinach test portions each. The first round yielded ∼84% and ∼82% positive rates across laboratories for the qPCR and culture methods, respectively, which were both outside the fractional range (25%-75%) required for fractionally inoculated test portions by the FDA's Microbiological Method Validation Guidelines. The second round yielded ∼68% and ∼67% positive rates. The relative level of detection (RLOD) for the second-round study was 0.969, suggesting that qPCR and culture methods had similar sensitivity (p > 0.05). The study demonstrated that the qPCR yields reproducible results and is sufficiently sensitive and specific for the detection of Salmonella in food.

Successful Detection of Delta and Omicron Variants of SARS-CoV-2 by Veterinary Diagnostic Laboratory Participants in an Interlaboratory Comparison Exercise.

BACKGROUND: Throughout the COVID-19 pandemic, veterinary diagnostic laboratories have tested diagnostic samples for SARS-CoV-2 both in animals and over 6 million human samples. An evaluation of the performance of those laboratories is needed using blinded test samples to ensure that laboratories report reliable data to the public. This interlaboratory comparison exercise (ILC3) builds on 2 prior exercises to assess whether veterinary diagnostic laboratories can detect Delta and Omicron variants spiked in canine nasal matrix or viral transport medium. METHODS: The ILC organizer was an independent laboratory that prepared inactivated Delta variant at levels of 25 to 1000 copies per 50 µL of nasal matrix for blinded analysis. Omicron variant at 1000 copies per 50 µL of transport medium was also included. Feline infectious peritonitis virus (FIPV) RNA was used as a confounder for specificity assessment. Fourteen test samples were prepared for each participant. Participants used their routine diagnostic procedures for RNA extraction and real-time reverse transcriptase-PCR. Results were analyzed according to International Organization for Standardization (ISO) 16140-2:2016. RESULTS: Overall, laboratories demonstrated 93% detection for Delta and 97% for Omicron at 1000 copies per 50 µL. Specificity was 97% for blank samples and 100% for blank samples with FIPV. No differences in Cycle Threshold (Ct) values were significant for samples with the same virus levels between N1 and N2 markers, nor between the 2 variants. CONCLUSIONS: The results indicated that all ILC3 participants were able to detect both Delta and Omicron variants. The canine nasal matrix did not significantly affect SARS-CoV-2 detection.

Second round of an interlaboratory comparison of SARS-CoV2 molecular detection assays used by 45 veterinary diagnostic laboratories in the United States.

The COVID-19 pandemic presents a continued public health challenge. Veterinary diagnostic laboratories in the United States use RT-rtPCR for animal testing, and many laboratories are certified for testing human samples; hence, ensuring that laboratories have sensitive and specific SARS-CoV2 testing methods is a critical component of the pandemic response. In 2020, the FDA Veterinary Laboratory Investigation and Response Network (Vet-LIRN) led an interlaboratory comparison (ILC1) to help laboratories evaluate their existing RT-rtPCR methods for detecting SARS-CoV2. All participating laboratories were able to detect the viral RNA spiked in buffer and PrimeStore molecular transport medium (MTM). With ILC2, Vet-LIRN extended ILC1 by evaluating analytical sensitivity and specificity of the methods used by participating laboratories to detect 3 SARS-CoV2 variants (B.1; B.1.1.7 [Alpha]; B.1.351 [Beta]) at various copy levels. We analyzed 57 sets of results from 45 laboratories qualitatively and quantitatively according to the principles of ISO 16140-2:2016. More than 95% of analysts detected the SARS-CoV2 RNA in MTM at ≥500 copies for all 3 variants. In addition, for nucleocapsid markers N1 and N2, 81% and 92% of the analysts detected ≤20 copies in the assays, respectively. The analytical specificity of the evaluated methods was >99%. Participating laboratories were able to assess their current method performance, identify possible limitations, and recognize method strengths as part of a continuous learning environment to support the critical need for the reliable diagnosis of COVID-19 in potentially infected animals and humans.