Purpose

TaqPath SARS-CoV-2 PCR is a qualitative real-time PCR test designed to detect the novel SARS coronavirus 2 in respiratory samples. In this verification summary, we report our findings during performance characteristics analysis of ThermoFisher Scientific’s TaqPath COVID-19 Combo Kit. TaqPath™ COVID-19 Combo Kit contains the assays and controls for a real-time reverse transcription polymerase chain reaction (RT-PCR) test intended for the qualitative detection of nucleic acid from SARS-CoV-2 in upper respiratory specimens (such as nasopharyngeal, oropharyngeal, nasal, and mid-turbinate swabs, and nasopharyngeal aspirate), bronchoalveolar lavage (BAL), and saliva specimens from individuals suspected of COVID-19 by their healthcare provider.

TaqPath™ COVID-19 Combo Kit is for use only under Emergency Use Authorization (EUA).

Results are for the identification of SARS-CoV-2 RNA. The SARS-CoV-2 RNA is generally detectable in upper respiratory and bronchoalveolar lavage (BAL) specimens during the acute phase of infection.

Positive results are indicative of the presence of SARS-CoV-2 RNA. Negative results do not preclude SARS-CoV-2 infection and should not be used as the sole basis for patient management decisions. Negative results must be combined with clinical observations, patient history, and epidemiological information.

The workflow begins with nucleic acid extraction from upper respiratory specimens (such as nasopharyngeal, oropharyngeal, nasal, and mid-turbinate swabs, and nasopharyngeal aspirate) and bronchoalveolar lavage (BAL) specimens that arrive in the testing site in transport media. Nucleic acids are isolated and purified from the specimens using the MagMAX™ Viral/Pathogen Nucleic Acid Isolation Kit or the MagMAX™ Viral/Pathogen II Nucleic Acid Isolation Kit. Nucleic acid isolation can be performed manually or via an automated process using the KingFisher™ Flex Purification System (KingFisher). The purified nucleic acid is reverse transcribed into cDNA and amplified using the TaqPath™ RT-PCR COVID-19 Kit and QuantStudio 12K Flex. In the process, the probes anneal to three (3) specific SARS-CoV-2 target sequences located between three (3) unique forward and reverse primers for the following genes:

During the extension phase of the PCR cycle, the 5’ nuclease activity of Taq polymerase degrades the probe, causing the reporter dye to separate from the quencher dye, generating a fluorescent signal. With each cycle, additional reporter dye molecules are cleaved from their respective probes, increasing the fluorescence intensity. Fluorescence intensity is monitored at each PCR cycle by the real-time PCR instrument. The data are analyzed, then interpreted by the Applied Biosystems™ COVID‑19 Interpretive Software.

We purchased Zeptometrix purified, intact viral particles (NATtrol™ SARS-CoV-2 Stock and NATtrol™ SARS-CoV-2 External Run Controls in order to produce contrived samples for our experiments. The virus particles in NATtrol™ SARS-CoV-2 Stock and NATtrol™ SARS-CoV-2 External Run Controls have been chemically modified to render them non-infectious and refrigerator stable. The stock contains 1 million copies per mL of viral RNA and the external control contains 50,000 copies per mL of viral RNA. We used the stock to generate samples for our LoD studies and to generate high positive contrived samples. We used the external control to generate low positive contrived samples. Combination of nuclease-free water and nasopharyngeal swab preservative was used to generate negative samples. Details of specimen preparation are presented in Table 1.

The LoD was determined using Zeptometrix’s purified, intact viral particles (NATtrol™ SARS-CoV-2 Stock) that were diluted in SARS-CoV-2 negative nasopharyngeal swab matrix. The stock (106 copies/mL) was diluted 8 times in 1:10 steps (105 to 0.01 copies/mL). Each generated sample was then extracted and run in triplicate. Details of specimen generation for LoD studies have been presented in Table 1.
The lowest level at which all three replicates were positive for two of SARS-CoV-2 targets was 10 copies/mL. The estimated LoD was confirmed by testing an additional 20 replicates at the same target level. All 20 replicates produced the expected results for each SARS-CoV-2 target, and the LoD was therefore confirmed to be 10 copies/mL. The results are presented in Table 2.

Degree of variation from the mean Ct values was calculated for MS2 internal control within and between all the experiments. Standard deviations and %CVs were all within the acceptable error size and no imprecision was found. Results are presented in Table 3.

Contrived specimens were generated by spiking Zeptometrix inactivated viral particles or external control RNA in remnant negative nasopharyngeal samples. Negative samples were obtained from AmeriHealth Laboratory and included 15 nasopharyngeal samples (1 mL Puritan NP UTM swab) from previously tested patients who had no respiratory pathogens detected. We generated a group of high positive (105 copies/mL, n=15), and a group of low positive (103 copies/mL, n=15) contrived samples by spiking control material in remnant negative patient samples.

A negative group was generated (n=30) by adding nuclease-free water to the UTM medium. The contrived specimens were extracted, and PCR was run per protocol. Our assay detected the target genes 100% of the time (100% Positive Agreement). Our negative samples did not show any target detection for Orf1ab and S genes (100% negative agreement). We had amplification of N gene in 2 of our negative samples which are potential carry over contaminations from previous experiments. For N gene, our assay showed 93% negative agreement. Table 4 details the findings in our clinical evaluation/accuracy studies.

Inclusivity was performed in-silico by ThermoFisher Scientific as part of the TaqPath COVID-19 Combo Kit’s EUA submission. The assays were mapped to 185 complete SARS-CoV-2 genomes of human host in GenBank and GISAID databases as of March 5, 2020. Primer and probes sequences for SARS-CoV-2 ORF1ab, S gene, and N gene assays had 100% homology to all SARS-CoV-2 isolates analyzed, with one exception. EPI_ISL_407084 (Beta Coronavirus/Japan/AI/I-004/2020) showed a mismatch at position 7 from the 5’ end of the reverse primer (23 nt length) corresponding to 95.6% homology. The mismatch is located at the 5’end of the primer and does not affect the test performance.

Interference studies were performed by ThermoFisher Scientific as part of the TaqPath COVID-19 Combo Kit’s EUA submission: SARS-CoV-2-negative nasopharyngeal swab and bronchoalveolar lavage specimens were spiked with purified SARS-CoV-2 viral RNA at 3X the Limit of Detection (30 GCE/reaction) and potential interfering substances at the concentrations above. Each substance was tested with triplicate extractions. The results are presented in Table 5. Pooled SARS-CoV-2-negative nasopharyngeal swab and bronchoalveolar lavage specimens were spiked with potential interfering substances at the concentrations above. Each substance was tested with triplicate extractions. No false positive results were observed for any of the substances at the concentrations tested.