Given the scale of the ongoing COVID-19 pandemic, the need for reliable, scalable testing, and the likelihood of reagent shortages, especially in resource-poor settings, we have developed an RT-qPCR assay that relies on an alternative to conventional viral reverse transcriptases, a thermostable reverse transcriptase/DNA polymerase (RTX) (Ellefson et al., 2016). Here we show that RTX performs comparably to the other assays sanctioned by the CDC and validated in kit format. We demonstrate two modes of RTX use – (i) dye-based RT-qPCR assays that require only RTX polymerase, and (ii) TaqMan RT-qPCR assays that use a combination of RTX and Taq DNA polymerases (as the RTX exonuclease does not degrade a TaqMan probe). We also provide straightforward recipes for the purification of this alternative reagent RTX. We anticipate that in low resource or point-of-need settings researchers could obtain the available constructs and begin to develop their own assays, within whatever regulatory framework exists for them. While various virus detection methods have been implemented for the detection of SARS-CoV-2 infection, including a variety of molecular diagnostics and immunodiagnostic tests, the Reverse Transcriptase quantitative Polymerase Chain Reaction (RT-qPCR) remains the primary and most sensitive test for SARS-CoV-2 detection (D'Cruz et al., 2020; Tang et al., 2020). The primacy of RT-qPCR is in large measure because antibody-based tests as
well as rapid nucleic acid diagnostic platforms, such as Abbott IDNow, often suffer from poor sensitivity, especially during early infection when viral loads are generally low in patients (Basu et al., 2020; D'Cruz et al., 2020). Given the importance of the early diagnosis in containing COVID-19 outbreak (Peck, 2020), the need for a rapid and scalable RT-qPCR setup is imminent. Unfortunately, there are increasing shortages of a wide variety of reagents necessary to scale
RT-qPCR-based tests (Pettit et al., 2020). The main manufacturers of PCR platforms cannot scale-up the production of tests in sufficient quantities to supply resource-poor settings. Even were resource-poor settings to attempt to develop their own solutions, researchers, clinicians, and public health officials often lack the necessary reagents, including enzymes, to develop testing programs (Kavanagh et al., 2020).
Materials and Reagents
Equipment
Software
Procedure
Data analysis Analyze RT-qPCR data using LightCycler96 software. Example data for dye-based RTX-only RT-qPCR Representative results of SARS-CoV-2 RT-qPCR tests performed using synthetic RNA templates and only RTX or RTX Exo- DNA polymerases are depicted in Figures 1 and 2. These results demonstrate that RTX DNA polymerase alone, with or without proofreading capability, can support dye-based RT-qPCR analyses. In our hands, the full-length RTX DNA polymerase was slightly better than the Exo- version, especially in the N3 assay. Figure 1. CDC SARS-CoV-2 N1, N2, and N3 RT-qPCR assays performed using indicated copies of synthetic RNA and RTX DNA polymerase. Panels A, B, and C depict N1, N2, and N3 assays measured in real-time using EvaGreen dye. Amplification curves from reactions containing 100,000 (black traces), 10,000 (blue traces), 1,000 (red traces), and 100 (pink traces) copies of SARS-CoV-2 synthetic N RNA are depicted. Negative control reactions either contained no templates (gray traces) or contained 100,000 copies of synthetic N RNA from MERS-CoV (green traces). Panels D, E, and F depict melting peaks of amplicons determined using the ‘Tm calling’ analysis in the LightCycler96 software. Note that the amplicons observed in negative controls were spurious, as indicated by their distinct melting temperatures compared to target-derived amplicons. Average Cq values of all assays are tabulated. Figure 2. CDC SARS-CoV-2 N1, N2, and N3 RT-qPCR assays performed using indicated copies of synthetic RNA and RTX Exo- DNA polymerase. Panels A, B, and C depict N1, N2, and N3 assays measured in real-time using EvaGreen dye. Amplification curves from reactions containing 100,000 (black traces), 10,000 (blue traces), 1,000 (red traces), and 100 (pink traces) copies of SARS-CoV-2 synthetic N RNA are depicted. Negative control reactions either contained no templates (gray traces) or contained 100,000 copies of synthetic N RNA from MERS-CoV (green traces). Panels D, E, and F depict melting peaks of amplicons determined using the ‘Tm calling’ analysis in the LightCycler96 software. Average Cq values of all assays are tabulated. Representative results of RTX polymerase-based SARS-CoV-2 TaqMan RT-qPCR tests performed using synthetic RNA templates are depicted in Figures 3 and 4. Both RTX and RTX Exo- when combined with Taq DNA polymerase are able to support one-pot TaqMan RT-qPCR assays for all three CDC SARS-CoV-2 assays. Under both buffer conditions tested, 1x ThermoPol versus 1x RTX buffer, RTX polymerase yielded more consistent amplification curves for all three CDC assays compared to RTX Exo- polymerase. Figure 3. CDC SARS-CoV-2 N1, N2, and N3 TaqMan RT-qPCR assays performed in 1X ThermoPol buffer using indicated copies of synthetic RNA and RTX or RTX Exo- and Taq DNA polymerases.Panels A, B, and C depict TaqMan assays containing both RTX and Taq DNA polymerases. Panels D, E, and F depict TaqMan assays containing only Taq DNA polymerase. Panels G, H, and I depict TaqMan assays containing RTX Exo- and Taq DNA polymerases. Amplification curves from reactions containing 100,000 (black traces), 10,000 (blue traces), 1,000 (red traces), and 100 (pink traces) copies of SARS-CoV-2 synthetic N RNA are depicted. Negative control reactions either contained no templates (gray traces) or contained 100,000 copies of synthetic N RNA from MERS-CoV (green traces). Cq values of all assays are tabulated.
Figure 4. CDC SARS-CoV-2 N1, N2, and N3 TaqMan RT-qPCR assays performed in 1X RTX buffer using indicated copies of synthetic RNA and RTX or RTX Exo- and Taq DNA polymerases. Panels A, B, and C depict TaqMan assays containing RTX and Taq DNA polymerases. Panels D, E, and F depict TaqMan assays containing RTX Exo- and Taq DNA polymerases. Amplification curves from reactions containing 100,000 (black traces), 10,000 (blue traces), 1,000 (red traces), and 100 (pink traces) copies of SARS-CoV-2 synthetic N RNA are depicted. Negative control reactions either contained no templates (gray traces) or contained 100,000 copies of synthetic N RNA from MERS-CoV (green traces). Cq values of all assays are tabulated. Example data for TaqPathTM TaqMan RT-qPCR Figure 5 depicts typical results from CDC N1, N2, and N3 TaqMan qRT-PCR reactions performed using synthetic RNA and TaqPathTM commercial RT-qPCR mastermix. Figure 5. CDC SARS-CoV-2 N1, N2, and N3 TaqMan RT-qPCR assays performed using indicated copies of synthetic RNA and TaqPathTM commercial RT-qPCR mastermix. Amplification curves from reactions containing 100,000 (black traces), 10,000 (blue traces), 1,000 (red traces), and 100 (pink traces) copies of SARS-CoV-2 synthetic N RNA are depicted. Negative control reactions either contained no templates (gray traces) or contained 100,000 copies of synthetic N RNA from MERS-CoV (green traces). Cq values of all assays are tabulated. The results with either a viral RT (in TaqPathTM commercial mastermix) or RTX are summarized in Figure 6. As can be seen, RTX-based TaqMan assays are of comparable sensitivity and specificity to the gold standard TaqPath assay. While all three RTX-based TaqMan assays performed in ThermoPol buffer consistently yielded Cq values, these were somewhat delayed compared to Cq values obtained with the commercial TaqPathTM Master Mix. In contrast, RTX-based TaqMan assays when executed in RTX buffer yielded Cq values that were closer to Cq values obtained with the commercial mastermix. Figure 6. Comparison of Cq values of RTX-based and TaqPath-based SARS-CoV-2 TaqMan RT-qPCR assays
Figure 7. An example of RTX purification analyzed by SDS-PAGE. A total of ten to forty micrograms of purified RTX was developed on a NuPAGETM 4 to 12%, Bis-Tris mini protein gel. RTX bands appear near the 100kDa size marker. 1st lane: 10 µg, 2nd lane: 20 µg, 3rd lane: 30 µg, 4th lane: 40 µg. Notes Overall, RTX performs well either as a single enzyme for RT-qPCR with intercalating dyes, such as EvaGreen, or as the RT component of a TaqMan based assay. At the lowest RNA concentrations examined (100 copies), the gold standard TaqPath assay generally gave a signal at a Cq value of ca. 32. The more robust full-length RTX on its own actually performed slightly better, with Cq values typically from 29-30 (although the N3 primer set gives a higher signal and higher background). When used as a substitute for a viral RT, RTX is slightly less sensitive overall, with Cq values typically from 32-35. By obtaining a RTX expression vector and purifying the thermostable reverse transcriptase it should prove possible to carry out RT-qPCR reactions with a sensitivity similar to that already observed for approved kits, with few or no false positive results. Plasmids and sequence information for 6xHis tagged RTX and RTX Exo- protein expression are available from Addgene (pET_RTX: https://www.addgene.org/102787/ and pET_RTX_(exo-): https://www.addgene.org/102786/), or can be obtained via https://reclone.org/. Recipes
Buffers for RTX DNA polymerase purification
Buffers for dialysis of RTX DNA polymerase
Acknowledgments We acknowledge the extraordinary contributions of the Schoggins lab at the University of Texas Southwestern Medical Center in making synthetic RNA available on short notice for these assays. We would like to acknowledge funding from the Promega Corporation (UTA18-000656), the National Institutes of Health (1R01EB027202-01A1), and the Welch Foundation (F-1654). Competing interests The Board of Regents of The University of Texas has licensed IP covering RTX to Promega Corporation. References
Copyright: © 2021 The Authors; exclusive licensee Bio-protocol LLC. How to cite: Bhadra, S., Maranhao, A. C., Paik, I. and Ellington, A. D. (2021). A
One-enzyme RT-qPCR Assay for SARS-CoV-2, and Procedures for Reagent Production. Bio-protocol 11(2): e3898. DOI: 10.21769/BioProtoc.3898. |