IDEXX SARS-CoV-2 (COVID-19) RealPCR Test
Analytical validation and cross-specificity studies
IDEXX Reference Laboratories developed a novel real-time PCR test to detect SARS-CoV-2 (COVID-19) based on a unique alignment to the published genetic sequences of the virus from the human outbreak. The IDEXX SARS-CoV-2 (COVID-19) RealPCR Test met all analytic validation requirements. Specificity studies showed no cross-reactivity with the new PCR test against common veterinary coronaviruses affecting companion animals. Likewise, currently available RealPCR tests for these veterinary coronaviruses were demonstrated to not detect synthetic COVID-19 nucleic acid. IDEXX RealPCR tests are designed and performed on a standard real-time PCR platform in the West Sacramento, California, molecular diagnostics (PCR) laboratory. More than 3,500 canine, feline, and equine specimens were screened with the new IDEXX SARS-CoV-2 (COVID-19) RealPCR Test. Patient samples were submitted over a four-week period beginning February 14, 2020, from across the United States and South Korea and included specimens from regions, such as the Seattle area, currently experiencing human COVID-19 cases. Specimens were also tested in parallel with three assays from the Centers for Disease Control and Prevention (CDC). In mid-March, IDEXX expanded monitoring to Canada and European countries, including areas with high rates of COVID-19 in the human population. By mid-April, over 5,000 specimens had been tested for the SARS-CoV-2 virus. All specimens have been negative.
Assay design and analytical validation
The IDEXX SARS-CoV-2 (COVID-19) RealPCR Test targets the same nucleocapsid gene as used in CDC assays. This target was based on the sequence analysis showing high conservation and adapted to meet the melting point and analytical requirements of our standardized platform. Validation of the test first involved sequence blast analysis against more than 300 publicly available genetic sequences. Second, a synthetic positive control was used to functionally validate the assay for performance, which met requirements for robust signal to noise during amplification efficacy, intra-run and inter-run reproducibility, and correlation. In addition, IDEXX implemented three CDC assays to run in parallel with the IDEXX assay for confirmatory purposes. The three confirmatory assays were adopted from the CDC, originally published on January 24, 2020.1,2
The cross-specificity of all four assays (SARS-COV-2) was confirmed using synthetic-positive veterinary coronavirus targets and PCR-positive canine respiratory coronavirus, canine and feline enteric coronavirus, and equine enteric coronavirus specimens submitted through the IDEXX diagnostic offering. The following controls ran during the real-time PCR validation phase and screening:
- Negative extraction control (NEC): all negative extraction controls scored negative by PCR.
- Preanalytical specimen quality control using a real-time TaqMan® PCR assay for an endogenous control gene. All specimens, regardless of negative or positive character, passed the specimen quality control.
- PCR negative controls: all PCR-negative controls scored negative.
- PCR positive controls: all PCR-positive controls (synthetic-positive control) tested positive.
- Environmental contamination monitoring: all specimens from different location within the PCR laboratory tested negative on the contamination monitoring
The PCR-positive canine respiratory coronavirus, canine and feline enteric coronavirus, and equine enteric coronavirus specimens, which were submitted though the IDEXX diagnostic offering, were also screened against all SARS-CoV-2 assays. All CDC assays and IDEXX SARS-CoV-2 (COVID-19) RealPCR assays tested negative on this population of other coronaviruses.
Randomly selected specimens from the specimens submitted through the test system of IDEXX RealPCR for canine respiratory, feline respiratory, equine respiratory, feline diarrhea, canine diarrhea, and equine diarrhea were used. Initial specimens used for screening included swabs (majority are deep pharyngeal [throat] and conjunctival [eye] swabs) and feces. Of the more than 3,500 specimens screened from February 14 to March 12:
- Specimens screened were from all 50 states and from South Korea.
- 55% of the specimens were from canine, 41% were from feline, 4% were from equine.
- 77% were respiratory, 23% were fecal.
- Centers for Disease Control and Prevention, Division of Viral Diseases. CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel Instructions for Use. CDC publication CDC-006-00019, Revision: 02. www.fda.gov/media/134922/download. Effective March 15, 2020. Accessed March 16, 2020.
- Centers for Disease Control and Prevention, Division of Viral Diseases. Research Use Only Real-Time RT-PCR Protocol for Identification of 2019-nCoV. www.cdc.gov/coronavirus/2019-ncov/lab/rt-pcr-detection-instructions.html. Updated March 14, 2020. Accessed March 16, 2020.