A total of 300 randomly selected nasopharyngeal specimens were evaluated in this study that were collected from patients suspected of having COVID-19 infection between May and June of 2020. Specimens were transported to the laboratory in Universal Transport Medium (UTM) and tested using the Xpert Xpress SARS-CoV-2 assay within 2 hours of collection. Of the 300 specimens, 60 were positive and 240 were negative for SARS-CoV-2 by the Xpert Xpress assay. The specimens were frozen at -70°C immediately after testing. Specimen were then transported on dry ice to the Masonic Medical Research Institute located in Utica, NY where they were batch tested using the TaqPath COVID-19 Combo Kit. All specimens underwent only one freeze thaw cycle prior to TaqPath testing.
Ethical Approval and Consent to Participate
The study was conducted using residual, de-identified specimens and no clinical or demographic information was collected. The study was conducted in accordance with the FDA guidance on Informed Consent for In Vitro Diagnostic Device Studies Using Leftover Human Specimens that are Not Individually Identifiable (April 2006). This guidance advises that informed consent is not required for this study design. The protocol was reviewed and approved by the Advarra Institutional Review Board (Columbia, MD) and determined that informed consent was not required for this study (CR0025653).
Xpress SARS-CoV-2 Assay
The Xpert Xpress SARS-CoV-2 assay is an automated in vitro diagnostic test for the qualitative detection of SARS-CoV-2 RNA using reverse transcription-PCR (RT-PCR). The Xpert Xpress SARS-CoV-2 assay is performed within a self-contained cartridge that performs extraction, amplification, and detection of amplicons if the target gene(s) are present. The cartridge also contains a Sample Process Control (SPC) and a Probe Check Control (PCC). The SPC controls for the adequate processing of the specimen and monitors for the presence of potential inhibitor(s) to the RT-PCR reaction. The PCC verifies reagent rehydration and monitors other functional activities within the cartridge.
The Xpress SARS-CoV-2 assay was performed according to the manufacturer’s instructions [10]. Basically, the specimen in UTM was mixed by inversion 5 times, a 300 µL volume was transferred to the test cartridge, and the cartridge was loaded into the Gene Xpert instrument. The assay targets the N2 and E gene sequences and, according to the manufacturer, has a LoD of 250 copies/mL. The assay has a crossing threshold (Ct) cut-off value of ≥45 cycles for negative specimens and is completed within 50 minutes.
TaqPath Combo Kit Assay
The TaqPath Combo Kit is a high complexity assay that requires a separate, stand-alone nucleic acid extraction step. The assay is performed using a 96 well microtiter tray that allows for the testing of 94 specimens as well as a positive and negative control per run. Gene amplification and amplicon detection can be performed by using any one of a number of instrument platforms, such as Applied Biosystems 7500 Fast Dx, as was used in this study. The assay targets 3 gene sequences, (N2, ORF1ab, and S genes) and is completed within 3 hours.
The assay was performed according to the manufacturer’s instructions [11] by first extracting a 400 µL aliquot of specimen in UTM using the MagMAX™ Viral/Pathogen Nucleic Acid isolation kit on the KingFisher Flex Purification system (Thermo Fisher Scientific, Waltham, MA). Prior to RNA extraction, 10µL of Proteinase K was added to each well in the KingFisher™ Deepwell 96 Plate. In addition, 10µL of the MS2 Phage Control was added to all specimens and the Negative Control that served as an internal process control. The nucleic acid was eluted into 50µL of Elution Solution.
For each specimen, Master Mix was prepared containing TaqPath 1-Step Multiplex Master Mix (No ROX™), COVID-19 Real Time PCR Assay Multiplex, and Nuclease-free water. 20µL of Master Mix was dispensed into wells in a 96 well plate followed by the addition of 5µL of eluted specimen to the appropriate well. Each run also included a SARS-CoV-2 Positive Control and a Negative Control.
Amplification was performed on the Applied Biosystems® 7500 Fast Dx Real-Time PCR Instrument (ThermoFisher Scientific, Waltham, MA). Testing was performed in batches of 94 specimens plus one negative and positive control. The results were interpreted using the Applied Biosystems™ COVID-19 Interpretive Software version 1.3. According to the manufacturer’s instructions, a specimen was considered SARS-CoV-2 positive when 2 or more SARS-CoV-2 gene targets were called positive with cycle threshold values of ≤37. The time to complete the assay for 94 specimens including the controls was approximately 3 hours. According to the manufacturer, the assay has an LoD of 250 copies/mL.
Discordant Specimen Testing
Specimens that produced discordant test results between the Xpress and TaqPath assays were arbitrated using two different methods: QIAstatDx™ Respiratory Panel (Qiagen, Germantown, MD) and Sanger sequencing. The QIAstatDx Respiratory Panel is a qualitative, multiplex assay that simultaneously screens for over 20 respiratory pathogens, including SARS-CoV-2. For SARS-CoV-2, the assay targets the ORF1b and E gene sequences. The assay which was performed according to the manufacturer’s instructions [12] is completed in approximately 1 hour and has a LoD of 500 copies/mL. The Ct cutoff for a negative specimen is ≥37 cycles.
Sanger Sequencing Method
Specimens that were discordant between the Xpert Xpress and TaqPath methods were also arbitrated using Sanger sequencing. Specimens were preamplified using a cocktail of N2, ORF1ab, and S primers that flanked the regions analyzed using the TaqPath COVID-19 Multiplex Diagnostic Solution. Five microliters of specimen were combined with 2.5µl 4x TaqPath™ 1-Step RT-qPCR Master Mix, 1µl preamp primer cocktail (final concentration 100 nM each primer), and 1.5µl water (10µl total volume). Specimens were preamplified using the following PCR profile: 15 min 37°C, 95°C 2 min, then 25 cycles of 95°C 3 sec, 60°C 45 sec, a single final 5 min extension of 72°C and 4°C hold. Following preamplification, 4µl of ExoSAP-It™ was added to the tube and incubated at 37°C for15 min, then 80°C for 7 min to remove the preamplification primers.
The preamplified specimens were sequenced using the Big Dye Direct protocol. Forward and reverse primers for the N2, ORF1ab, and S genes were designed with M13 forward and reverse sequencing tags. Sequencing amplicons were generated by combining 1µl preamp reaction, 5µl 2x Big Dye Direct PCR master mix, 1µl N2, ORF1ab, or S M13-tagged amplification pair (final concentration 100nM). Each reaction was set up in duplicate. Amplification was performed using the following protocol: 10 min 95°C, then 40 cycles of 96°C 3 sec, 62°C 15 sec 68°C 30 sec, and 4°C hold. Following PCR amplification, 2µl Big Dye Direct Sequencing master mix and 1µl of either Big Dye Direct M13 Forward or Reverse primer were added. Cycle sequencing was performed using the protocol: 15 min 37°C, 80°C 2 min, 95°C 1 min, then 25 cycles of 96°C 10 sec, 50°C 5 sec, 65°C 75 sec, and 4°C hold. Sequencing reactions were cleaned using the Big Dye Xterminator according to instructions supplied with the kit and analyzed on Applied Biosystems 3500xl instrument with 50 cm capillary and POP7 polymer.
Sequencing traces were analyzed using Applied Biosystems SeqScanner 2. QC Reports were generated for each trace. A trace passed if two of these three criteria was met: Trace score >31, Contiguous read length (CRL) >50, and QV20+ score >50. Sequences were also aligned with the SARS-CoV-2 genome to verify identity. A specimen was called positive for the viral genome if at least one passable trace in each amplicon, in either direction, was present.