A blueprint for academic laboratories to produce SARS-CoV-2 quantitative RT-PCR test kits

Abstract

Widespread testing for the presence of the novel coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in individuals remains vital for controlling the COVID-19 pandemic prior to the advent of an effective treatment. Challenges in testing can be traced to an initial shortage of supplies, expertise, and/or instrumentation necessary to detect the virus by quantitative RT-PCR (RT-qPCR), the most robust, sensitive, and specific assay currently available. Here we show that academic biochemistry and molecular biology laboratories equipped with appropriate expertise and infrastructure can replicate commercially available SARS-CoV-2 RT-qPCR test kits and backfill pipeline shortages. The Georgia Tech COVID-19 Test Kit Support Group, composed of faculty, staff, and trainees across the biotechnology quad at Georgia Institute of Technology, synthesized multiplexed primers and probes and formulated a master mix composed of enzymes and proteins produced in-house. Our in-house kit compares favorably with a commercial product used for diagnostic testing. We also developed an environmental testing protocol to readily monitor surfaces for the presence of SARS-CoV-2. Our blueprint should be readily reproducible by research teams at other institutions, and our protocols may be modified and adapted to enable SARS-CoV-2 detection in more resource-limited settings.

Keywords: DNA polymerase; RNA; RT-qPCR; SARS-CoV-2; coronavirus; formulation; infectious disease; polymerase chain reaction (PCR); protein purification; reverse transcriptase; reverse transcription; ribonuclease inhibitor; virus.

Figure 1.

Project components and workflow.

Figure 2.

Performance of Georgia Tech multiplex primers and probes in several commercially available master mixes. Shown is GT multiplex primer/probe performance in commercial TaqPath, TaqPath Multiplex, and TaqMan Fast Virus 1-Step master mixes. Commercial master mix identity had no detectable impact on performance of the GT-made multiplex primer/probe mix. Due to the proximity of FAM and HEX channels, bleed-through from the FAM into the HEX channel was observed (see bottom nCov plasmid panels) but was of lower intensity than signal generated by the HEX-RP-BHQ1 probe (see top panels) and did not interfere with analyses when the HEX fluorescence threshold (blue dashed line) was set above the bleed-through intensity. Template in the top row consisted of synthetic SARS-CoV-2 RNA (ATCC) mixed with HEK293T RNA. Results are consistent with those expected for a positive patient sample. A negative sample would consist of a single amplification curve in the HEX channel (blue line). Template in the bottom row was 2019_nCoV_N_Positive Control (IDT) plasmid DNA.

Figure 3.

Performance of GT RT-qPCR Master Mix. RT-qPCR was performed with Georgia Tech thermal cycling conditions (Table 2) GT-Master Mix components (Table 4), using ATCC synthetic viral RNA (ATCC^® VR-3276SD™) template, and C_t values were determined using a threshold of 0.1, unless otherwise noted. A, effect of CHAPSO (0.1%) and BSA (0.5 mg/ml) on GT-Master Mix (GT-MM) performance with IDT N1 primers and 50,000 copies of synthetic viral RNA. The no-template control did not amplify. B, performance of GT multiplex primers and probes with 50,000 copies of viral RNA with GT-Master Mix, compared with TaqPath, and effect of trehalose (9.5%) added to GT-Master Mix. C, performance of GT-Master Mix with IDT N1 primers and 500 copies of synthetic viral RNA, compared with TaqPath, after three freeze/thaw cycles (6 days of storage) at 2× concentration. Inset, C_t for GT-Master Mix and TaqPath over 6 days of storage. D, qPCR efficiency (E = 10^(−1/slope) − 1) using autothreshold. GT-Master Mix and GT multiplex primers (N1 and N2 FAM readout, blue): 91.6%; GT-Master Mix and GT singleplex primers (brown): 87.8% for GT-N1 primer/probe (diamond), 77.1% for GT-N2 primer/probe (triangle), 86.4% for GT-RP primer/probe (inverted triangle), and 100.3% for TaqPath with GT-N1 primer/probe (red). Singleplex RT-qPCRs were performed with a mix of full-length viral RNA and HEK293T total RNA.

Figure 4.

Environmental testing protocol and qPCR standard curve. A, environmental testing protocol (see “Environmental testing”). B, standard curves used to calculate the magnitude of environmental surface contamination and qPCR efficiencies (E = 10 ^(−1/slope) − 1) using TaqPath and IDT CDC primers and probes. Left template, Quantitative Synthetic SARS-CoV-2 RNA (ATCC #VR-3276SD), N1 r² = 0.993, N2 r² = 0.994. Right template, positive control plasmid viral DNA (IDT, #10006625), N1 r²= 0.997, N2 r² = 0.992.