IDEXX SARS-CoV-2 (COVID-19) RealPCR Test
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 and clinical 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. Clinical validation included successful completion of proficiency testing, resulting in external quality-control assurance certification through the European group, INSTAND e.V. The IDEXX test demonstrated 100% sensitivity and specificity on the specimens included in the proficiency testing. IDEXX RealPCR tests are designed on a standarized real-time PCR platform. Initial validation was performed in the West Sacramento, California, molecular diagnostics (PCR) laboratory. Canine, feline, and equine specimens were screened with the new IDEXX SARS-CoV-2 (COVID-19) RealPCR Test. Over 3,500 patient specimens were submitted over a four-week period from February 14–March 12, 2020. The specimens used for test development and validation were obtained from specimens submitted to IDEXX Reference Laboratories for respiratory PCR panels. The specimens originated from the United States and South Korea, and included specimens from regions, such as the Seattle area, that were experiencing significant 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 6,000 specimens had been tested for the SARS-CoV-2 virus. All specimens tested 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 temperature 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 ratio, amplification efficiency, 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
Clinical validation and proficiency testing
Due to the extreme paucity of veterinary SARS-CoV-2 infections globally, clinical validation was performed using characterized clinical isolates from humans. 48 clinically characterized human isolates (32 SARS-CoV-2 positive and 16 negative) were tested and compared to results from an approved human diagnostic laboratory. All characterized SARS-CoV-2 positive specimens tested positive and all characterized negative specimens tested negative with the IDEXX SARS-CoV-2 (COVID-19) RealPCR Test.
IDEXX Reference Laboratories was also invited to participate in two rounds of proficiency testing through INSTAND e.V., a European group that provides certification in external quality-control assurance for diagnostic laboratories. This proficiency testing involved testing of characterized clinical specimens, including heat-inactivated specimens positive for SARS-CoV-2 in varying concentrations, and specimens positive for seasonal human CoV and MERS-CoV in varying concentrations. IDEXX achieved certification after demonstrating 100% sensitivity and specificity in detection of SARS-CoV-2 virus in these specimens.
The 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 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.
Cross-specificity of currently available IDEXX RealPCR tests for veterinary coronaviruses was also confirmed through both blast analysis and through 100% negative PCR results when tested against 55 clinically characterized human isolates (36 SARS-CoV-2 positive and 19 negative).
Specimens were selected at random from specimens submitted to IDEXX Reference Laboratories for IDEXX RealPCR canine respiratory, feline respiratory, canine diarrhea, and equine diarrhea panels. Initial specimens used for screening included swabs (majority were 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.
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 6,000 specimens had been tested for the SARS-CoV-2 virus. All specimens tested negative. Surveillance was discontinued on April 17, 2020.
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.
- 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.