2019 ncov cdc eua kit

A mix of 40 known-negative and 50 SARS-CoV-2 known-positive patient nasal swab samples were tested on the Franklin three9 using the Biomeme SARS-CoV-2 assay. Patient samples that were negative for COVID-19 also included ones where other respiratory viruses had been detected by conventional clinical assays. The samples were assessed using the relevant clinical gold-standard test where specimens were either extracted using the NucliSENS EasyMAG and subsequently run on the Rotor-gene Q, detecting the 5′UTR and envelope gene or were extracted and run on the BD Max 5′UTR assay, detecting only the 5′UTR gene^{11 (link)}. Additionally, negative patient samples were spiked with various clinical bacterial isolates to determine specificity. The clinical evaluation of the Precision Biomonitoring TripleLock SARS-CoV-2 Assay was conducted using 63 known-positive and 64 known-negative patient specimens. These samples were extracted using the NucliSENS EasyMAG kit and were confirmed positive or negative using a modified 2019-nCoV CDC EUA Kit (Integrated DNA Technologies, Coralville, USA) with the Luna Universal Probe One-Step RT kit (New England BioLabs, Whitby, Canada) and SARS-CoV-2 envelope gene assays^{11 (link)}. Extraction and RRT-PCR setup for the Biomeme and Precision Biomonitoring SARS-CoV-2 assays were carried out as described above for patient specimens.

The qPCR mixture with primers and probe was prepared for analysis of the CDC N2 amplicon, using N2 primer and probe (2019-nCov CDC EUA kit, product number 10006770; Integrated DNA Technologies) and Luna universal probe qPCR master mix (product number M3004; New England Biosystems). To analyze 96 samples, including controls, 300 μl of water and 200 μl N2 of primer/probe mixture were added to a tube containing 1 ml of 2× Luna probe mixture. A 96-well qPCR plate was placed on a cold block on ice, and 15 μl of this mixture was added to each well. Using a multichannel pipette, 5 μl of each swab eluate was added into a corresponding well of the plate containing the qPCR mix. On each plate, negative-control swabs, wells containing only TE buffer, and positive controls known to contain the N2 DNA amplicon were also analyzed. The plate was sealed with optical film and placed in a LightCycler 96 qPCR machine (Roche), which was run for 45 cycles. Data were analyzed using Prism 8 software (GraphPad). Values for samples with undetectable DNA (no cycle threshold [C_T] value) were set at a C_T of 45 for purposes of visualization.

The qPCR mixture with primers and probe was prepared for analysis of the CDC N2 amplicon, using N2 primer and probe (2019-nCov CDC EUA kit, product number 10006770; Integrated DNA Technologies) and Luna universal probe qPCR master mix (product number M3004; New England Biosystems). To analyze 96 samples, including controls, 300 μl of water and 200 μl N2 of primer/probe mixture were added to a tube containing 1 ml of 2× Luna probe mixture. A 96-well qPCR plate was placed on a cold block on ice, and 15 μl of this mixture was added to each well. Using a multichannel pipette, 5 μl of each swab eluate was added into a corresponding well of the plate containing the qPCR mix. On each plate, negative-control swabs, wells containing only TE buffer, and positive controls known to contain the N2 DNA amplicon were also analyzed. The plate was sealed with optical film and placed in a LightCycler 96 qPCR machine (Roche), which was run for 45 cycles. Data were analyzed using Prism 8 software (GraphPad). Values for samples with undetectable DNA (no cycle threshold [C_T] value) were set at a C_T of 45 for purposes of visualization.