A blueprint for academic laboratories to produce SARS-CoV-2 quantitative RT-PCR test kits
2020; Elsevier BV; Volume: 295; Issue: 46 Linguagem: Inglês
10.1074/jbc.ra120.015434
ISSN1083-351X
AutoresSamantha J. Mascuch, Sara Fakhretaha-Aval, Jessica C. Bowman, Minh Thu, Gwendell M. Thomas, Bettina Bommarius, Chieri Ito, Liangjun Zhao, Gary P. Newnam, Kavita Matange, Hem R. Thapa, Brett M. Barlow, Rebecca K. Donegan, Nguyet A. Nguyen, Emily Saccuzzo, Chiamaka T. Obianyor, Suneesh C. Karunakaran, Paméla Pollet, Brooke Rothschild-Mancinelli, Santi Mestre-Fos, Rebecca Guth-Metzler, Anton V. Bryksin, Anton S. Petrov, Mallory Hazell, Carolyn B. Ibberson, Petar I. Penev, Robert G. Mannino, Wilbur A. Lam, Andrés J. Garcı́a, Julia Kubanek, Vinayak Agarwal, Nicholas V. Hud, Jennifer B. Glass, Loren Dean Williams, Raquel L. Lieberman,
Tópico(s)Viral gastroenteritis research and epidemiology
ResumoWidespread 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. 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. The global COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) substantially disrupted activities in the public and private sectors (1Wu F. Zhao S. Yu B. Chen Y.-M. Wang W. Song Z.-G. Hu Y. Tao Z.-W. Tian J.-H. Pei Y.-Y. Yuan M.-L. Zhang Y.-L. Dai F.-H. Liu Y. Wang Q.-M. et al.A new coronavirus associated with human respiratory disease in China.Nature. 2020; 579 (32015508): 265-26910.1038/s41586-020-2008-3Crossref PubMed Scopus (7724) Google Scholar, 2Zhou P. Yang X.-L. Wang X.-G. Hu B. Zhang L. Zhang W. Si H.-R. Zhu Y. Li B. Huang C.-L. Chen H.-D. Chen J. Luo Y. Guo H. Jiang R.-D. et al.A pneumonia outbreak associated with a new coronavirus of probable bat origin.Nature. 2020; 579 (32015507): 270-27310.1038/s41586-020-2012-7Crossref PubMed Scopus (14613) Google Scholar, 3Zhu N. Zhang D. Wang W. Li X. Yang B. Song J. Zhao X. Huang B. Shi W. Lu R. Niu P. Zhan F. Ma X. Wang D. Xu W. et al.A novel coronavirus from patients with pneumonia in China, 2019.N. Engl. J. Med. 2020; 382 (31978945): 727-73310.1056/NEJMoa2001017Crossref PubMed Scopus (18869) Google Scholar). Widespread and frequent testing, in conjunction with contact tracing and behavioral change, has been demonstrated by some countries to be effective in monitoring and managing the outbreak. These strategies will continue to be instrumental in containing the virus until a vaccine or other effective treatment is universally available (4Patel R. Babady E. Theel E.S. Storch G.A. Pinsky B.A. George K.S. Smith T.C. Bertuzzi S. Report from the American Society for Microbiology COVID-19 international summit, 23 March 2020: value of diagnostic testing for SARS–CoV-2/COVID-19.mBio. 2020; 11 (32217609): e00720-e0072210.1128/mBio.00722-20Crossref Scopus (263) Google Scholar). Whereas comprehensive testing programs have been successfully implemented in many countries, testing efforts in the United States were hampered by a lack of access and an uncoordinated approach to early testing. The Georgia Tech COVID-19 Test Kit Support Group was conceived to leverage in-house Georgia Tech facilities, expertise, and personnel to assist the State of Georgia Clinical Laboratory Improvement Amendments (CLIA) laboratory with materials needed for clinical SARS-CoV-2 detection. Similar efforts are under way at several other universities (5Bhadra S. Maranhao A.C. Ellington A.D. One enzyme reverse transcription qPCR using Taq DNA polymerase.bioRxiv. 2020; 10.1101/2020.05.27.120238Google Scholar, 6Graham, T. G. W., Dailey, G. M., Dugast-Darzacq, C., and Esbin, M. N., (2020) BEARmix version 2: basic economical amplification reaction one-step RT-qPCR master mix. https://gitlab.com/tjian-darzacq-lab/bearmix/-/blob/b41ca99e563568527aace4a2e87cdad9417e2d9a/BEARmix_v4.pdf.Google Scholar), with some going so far as to establish "pop-up" testing laboratories (7Amen A.M. Barry K.W. Boyle J.M. Testing Consortium, IBlueprint for a pop-up SARS-CoV-2 testing lab.Nat. Biotechnol. 2020; 38 (32555529): 791-79710.1038/s41587-020-0583-3Crossref PubMed Scopus (33) Google Scholar). To our knowledge, ours is the first effort to use all in-house materials and equipment, offering an open-access community resource for other settings with similar capabilities. Much of the testing shortfall, especially early in the pandemic, can be traced to a shortage of reagents, plasticware, expertise, or instrumentation necessary to perform quantitative RT-PCR (RT-qPCR (8Holland P.M. Abramson R.D. Watson R. Gelfand D.H. Detection of specific polymerase chain reaction product by utilizing the 5'–3' exonuclease activity of Thermus aquaticus DNA polymerase.Proc. Natl. Acad. Sci. U. S. A. 1991; 88 (1871133): 7276-728010.1073/pnas.88.16.7276Crossref PubMed Scopus (2251) Google Scholar)). In RT-qPCR, RNA is converted to cDNA, which is then amplified via PCR until a detection threshold is reached. The TaqMan RT-qPCR method is widely considered the "gold standard" for SARS-CoV-2 testing due to its robustness, high sensitivity, linearity, and specificity (9Esbin M.N. Whitney O.N. Chong S. Maurer A. Darzacq X. Tjian R. Overcoming the bottleneck to widespread testing: a rapid review of nucleic acid testing approaches for COVID-19 detection.RNA. 2020; 26 (32358057): 771-78310.1261/rna.076232.120Crossref PubMed Scopus (359) Google Scholar). In a TaqMan RT-qPCR, the 5′–3′ exonuclease activity of a thermostable DNA polymerase cleaves a TaqMan oligonucleotide probe hybridized to the PCR amplicon. One terminus of the TaqMan probe is linked to a fluorophore, and the other terminus is linked to a quencher. Success in reverse transcription and PCR is detected as an increase in fluorescence upon probe cleavage during successive rounds of PCR, producing a sensitive and quantitative fluorescence signal that may be monitored in real time. A complete TaqMan RT-qPCR test kit includes (i) solution(s) of matched DNA probe(s) and primers specific to the gene target(s) of interest and (ii) an enzyme master mix. These solutions are mixed with a sample suspected to contain the RNA (e.g. SARS-CoV-2 RNA), run through a thermal cycling protocol in an RT-qPCR instrument, and monitored for an increase in fluorescence indicative of the presence of the target RNA. Solutions of matched probe and primer can be designed to detect one (singleplex) or multiple (multiplex) targets in a single reaction. Detection of a target usually needs to be differentiable from other targets in a multiplex reaction. In this case, a distinct fluorophore, with nonoverlapping emission wavelength, is used for each target. Commercial enzyme master mixes are sometimes branded for use with multiplex or singleplex primers and probes. The original CDC SARS-CoV-2 assay (10Lu X. Wang L. Sakthivel S.K. Whitaker B. Murray J. Kamili S. Lynch B. Malapati L. Burke S.A. Harcourt J. Tamin A. Thornburg N.J. Villanueva J.M. Lindstrom S. US CDC real-time reverse transcription PCR panel for detection of severe acute respiratory syndrome coronavirus 2.Emerg. Infect. Dis. 2020; 26 (32396505): 1654-166510.3201/eid2608.201246Crossref PubMed Scopus (392) Google Scholar) was a singleplex assay that required four distinct reactions for each sample suspected to contain the SARS-CoV-2 RNA: one for each of two sets of primers/probes (N1 and N2) targeting different regions of the N gene that encodes the SARS-CoV-2 nucleocapsid protein, one for a third set of primers/probe (N3) that detects all clade 2 and 3 viruses of the Betacoronavirus subgenus Sarbecovirus, and one for the primers/probe targeting human RNase P (RP). The latter is a control reaction for monitoring performance of the sample collection and RNA extraction. The CDC N3 primer/probe set was later eliminated due to template contamination and because it is unnecessary for specific detection of SARS-CoV-2 (10Lu X. Wang L. Sakthivel S.K. Whitaker B. Murray J. Kamili S. Lynch B. Malapati L. Burke S.A. Harcourt J. Tamin A. Thornburg N.J. Villanueva J.M. Lindstrom S. US CDC real-time reverse transcription PCR panel for detection of severe acute respiratory syndrome coronavirus 2.Emerg. Infect. Dis. 2020; 26 (32396505): 1654-166510.3201/eid2608.201246Crossref PubMed Scopus (392) Google Scholar, 11Willman D. Contamination at CDC lab delayed rollout of coronavirus tests.Washington Post. 2020; (May 18, 2020)Google Scholar), leaving three distinct reactions per sample. All probes in the CDC singleplex assay bear the common FAM fluorophore. Many companies subsequently developed FDA-approved multiplex SARS-CoV-2 primer/probe sets with a variety of fluorophores, enabling detection of all targets in a single reaction. Compared with singleplex, use of multiplex primer/probe sets substantially reduces the amount of enzyme mix and plasticware needed to process one patient sample but requires RT-qPCR instrumentation capable of monitoring the specific combination of fluorophores used. The TaqPath 1-Step RT-qPCR Master Mix (Thermo Fisher Scientific) was the first enzyme master mix to be recommended by the CDC and approved by the FDA for detection of SARS-CoV-2 (10Lu X. Wang L. Sakthivel S.K. Whitaker B. Murray J. Kamili S. Lynch B. Malapati L. Burke S.A. Harcourt J. Tamin A. Thornburg N.J. Villanueva J.M. Lindstrom S. US CDC real-time reverse transcription PCR panel for detection of severe acute respiratory syndrome coronavirus 2.Emerg. Infect. Dis. 2020; 26 (32396505): 1654-166510.3201/eid2608.201246Crossref PubMed Scopus (392) Google Scholar). TaqPath is a proprietary formulation containing a thermostable MMLV reverse transcriptase, a fast and thermostable DNA polymerase, an RNase inhibitor, a heat-labile uracil N-glycosylase (UNG), dNTPs including dUTP, ROX™ passive reference dye, and a buffer containing stabilizers and other additives. The DNA polymerase in TaqPath is likely to be a mutant of Taq polymerase incorporating some type of hot-start technology (12Green M.R. Sambrook J. Hot start polymerase chain reaction (PCR).Cold Spring Harbor Protoc. 2018; 2018 (29717052)10.1101/pdb.prot095125Google Scholar) to help suppress nonspecific amplification and primer dimers. UNG can remove carry-over contamination by specifically degrading products of prior PCRs that incorporate dUTP. Three other enzyme mixes, two made by Quantabio (qScript XLT One-Step RT-qPCR ToughMix (2×) and UltraPlex 1-Step ToughMix (4×)) and one made by Promega (GoTaq® Probe 1-Step RT-qPCR system) were added to the CDC list of master mix options shortly after TaqPath. The Quantabio mixes are provided as single components at 2× or 4× concentration, each containing a reverse transcriptase, antibody-based hot-start Taq DNA polymerase, RNase inhibitor protein, and the standard set of dNTPs. The Promega mix is provided as multiple components, including a 2× mix containing an antibody-based hot-start Taq and dNTPs (including dUTP) and a 50× mix containing reverse transcriptase and recombinant RNase inhibitor. Here we describe our in-house RT-qPCR assay for detection of SARS-CoV-2 (Fig. 1). First, we discuss preparation of singleplex and multiplex primers and probes with CDC sequences that can be used with commercial enzyme master mixes. Second, we present the production of reverse transcriptase (RT), Taq DNA polymerase, and RNase inhibitor (RI) proteins, and the formulation of a working one-step enzyme Georgia Tech master mix (GT-Master Mix) for use with our primers and probes. We compare the performance of our full in-house kit with that of a commercial kit. Finally, we describe implementation of environmental testing for SARS-CoV-2 across campus. We focused on producing the N1 and N2 primer and probe system published by CDC in March of 2020 (Table 1) because (i) these sequences had been extensively verified in the literature, (ii) our own bioinformatics analysis showed them to be highly specific to SARS-CoV-2 and localized to regions of the genome with low mutation rates (not shown), and (iii) they had received FDA Emergency Use Authorization (EUA). First, we synthesized and assayed the same singleplex primers and probes specified by the CDC. We then converted the CDC singleplex probes and primers to a multiplex system, in which N1 and N2 are detected via a common channel, and RNase P is detected in a separate channel. Specifically, the CDC FAM-RP-BHQ1 probe was converted to HEX-RP-BHQ1 to allow its simultaneous detection alongside FAM-N1-BHQ1 and FAM-N2-BHQ1, and the three probe/primer sets were combined in a single solution. The HEX fluorophore (maximum λem = 556 nm) is compatible with standard fluorophore channels of commercial RT-qPCR instruments and distinguishable from the FAM emission maximum at 518 nm. In addition, HEX has the second highest quantum yield (0.7) after FAM (0.9) and is commercially available. During development of our multiplex probe set, the OPTI SARS-CoV-2 RT-PCR multiplex test kit (13United States Food and Drug Administration (2020) OPTI SARS-CoV-2 RT-PCR test. https://www.fda.gov/media/137739/download.Google Scholar) which uses the same HEX-RP-BHQ1/FAM-N1-BHQ1/FAM-N2-BHQ1 probe configuration as our GT kit, gained EUA from the FDA (May 2020).Table 1Sequences of CDC primers and probesGene targetPrimer nameSequence and probe/quencher label (boldface)N (viral)2019-nCoV_N1-PFAM-ACCCCGCATTACGTTTGGTGGACC-BHQ12019-nCoV_N1-FGACCCCAAAATCAGCGAAAT2019-nCoV_N1-RTCTGGTTACTGCCAGTTGAATCTG2019-nCoV_N2-PFAM-ACAATTTGCCCCCAGCGCTTCAG-BHQ12019-nCoV_N2-FTTACAAACATTGGCCGCAAA2019-nCoV_N2-RGCGCGACATTCCGAAGAARP-P-FAMFAM-TTCTGACCTGAAGGCTCTGCGCG-BHQ1RNase P (human)RP-P-HEX1Substitute RP-P-HEX for RP-P-FAM in the multiplex reaction.HEX-TTCTGACCTGAAGGCTCTGCGCG-BHQ1RP-FAGATTTGGACCTGCGAGCGRP-RGAGCGGCTGTCTCCACAAGT1 Substitute RP-P-HEX for RP-P-FAM in the multiplex reaction. Open table in a new tab The GT Parker H. Petit Institute for Bioengineering and Bioscience's Molecular Evolution Core Facility dedicated its ASM-2000 high-throughput DNA/RNA synthesizer to primer and probe syntheses, which enabled tens of thousands of reactions worth of primers and probes to be produced in-house with a 6–8-h turnaround. Initially, HPLC was used to purify probes, but cross-contamination was detected from IDT positive control plasmid that was handled in the same laboratory (see details under "Environmental testing"). Learning from contamination issues faced by CDC (11Willman D. Contamination at CDC lab delayed rollout of coronavirus tests.Washington Post. 2020; (May 18, 2020)Google Scholar), and to further avoid potential contamination across the multitasking academic laboratory, a cartridge method was subsequently used to polish the FAM probes in the core facility, and HPLC was only used in another lab to analyze the purity of an aliquot of the material (Fig. S1, A–D). Primer purity was confirmed by gel electrophoresis after 32P end labeling (Fig. S1E). For the HEX probe, we used unpurified, but carefully synthesized HEX probe, based on the literature precedent that it should achieve similar efficiency as the high-purity probe (14Yeung A.T. Holloway B.P. Adams P.S. Shipley G.L. Evaluation of dual-labeled fluorescent DNA probe purity versus performance in real-time PCR.BioTechniques. 2004; 36 (14989091): 266-27510.2144/04362RR01Crossref PubMed Google Scholar). HEX probe purity ranged from 35 to 60% over different synthetic batches (not shown). The HEX probe exhibited temperature-dependent enhancement of fluorescence intensity, as expected (Fig. S1F). GT-made primers and probes generated robust and reproducible RT-qPCR signals with their respective targets. Prior to use with GT-Master Mix, primers and probes were individually validated by RT-qPCR to ensure acceptable performance in detecting N1, N2, and/or RP targets in commercial master mix (Fig. S2, A–C) and no contamination. Performance of FAM-labeled N1 and N2 is similar to that of commercial primers and probes purchased from IDT (not shown). The GT multiplex primers and probes mix was evaluated using several commercially available enzyme master mixes. The performance of multiplexed primers and probes did not differ among the enzyme master mixes tested (Fig. 2) and did not differ from that of the singleplex system (Fig. S2C). Thus, multiplexing did not impair enzyme function or deplete potentially limiting reagents, like dNTPs, from the master mix. Consistent with the greater quantum yield of FAM relative to HEX, probes that contained FAM (λabs = 494/λem = 518 nm) generated a greater ΔRn signal than did probes that contained HEX (λabs = 535/λem = 556 nm) in RT-qPCR experiments (Fig. S2C). Rn is the normalized reporter fluorescent dye signal normalized to the passive reference dye, and ΔRn is the Rn value of the experimental sample minus the instrument baseline signal. In fact, the spectral characteristics and intensity of the FAM signal were such that a portion of the signal could be observed in the adjacent HEX channel of the instrument, albeit at a much lower intensity ("bleed-through"; Fig. 2). The observed bleed-through of the FAM signal into the HEX channel likely arises due to spectral overlap of both HEX and FAM with the broad blue LED excitation (470/40 nm) in the StepOnePlus and QuantStudio 6 Flex instruments. Because the maximum ΔRn of the true HEX signal generated by the RP probe was always greater than the ΔRn from the bleed-through, unambiguous detection of HEX-RP-BHQ1 signal, indicating the presence of RNase P RNA in the sample, was possible by setting the HEX channel threshold above the ΔRn plateau of the bleed-through intensity (Fig. 2). To address this complication in a clinical setting, a simple MATLAB script was written to interpret the multiplex results in the context of bleed-through and provide a color-coded readout in Microsoft Excel (https://github.com/rmannino3/COVID19DataAnalysis). We obtained plasmids for two RT enzymes, an MMLV RT containing six mutations (15Arezi B. Hogrefe H. Novel mutations in Moloney murine leukemia virus reverse transcriptase increase thermostability through tighter binding to template-primer.Nucleic Acids Res. 2009; 37 (19056821): 473-48110.1093/nar/gkn952Crossref PubMed Scopus (67) Google Scholar) and RTX, an engineered xenopolymerase with proofreading activity (16Bhadra S. Maranhao A.C. Ellington A.D. A one-enzyme RT-qPCR assay for SARS-CoV-2, and procedures for reagent production.bioRxiv. 2020; 10.1101/2020.03.29/013342Google Scholar, 17Ellefson J.W. Gollihar J. Shroff R. Shivram H. Iyer V.R. Ellington A.D. Synthetic evolutionary origin of a proofreading reverse transcriptase.Science. 2016; 352 (27339990): 1590-159310.1126/science.aaf5409Crossref PubMed Scopus (95) Google Scholar). Both enzymes were purified to near homogeneity (Fig. S3, A and B) at high yield (10 mg/liter for GT-MMLV and 4 mg/liter for GT-RTX) by Ni2+-affinity chromatography followed by a second polishing step. RT activity was tested with in-house primers and templates from other projects (Fig. S3A) and remained highly active in RT-qPCR for ∼2.5 months; longer-term storage may require further optimization of the storage conditions. Characterization by OMNISEC reveals that GT-RTX is a dimer (167 kDa, Fig. S3B), compared with the monomeric MMLV RT (18Das D. Georgiadis M.M. The crystal structure of the monomeric reverse transcriptase from Moloney murine leukemia virus.Structure. 2004; 12 (15130474): 819-82910.1016/j.str.2004.02.032Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). In line with Bhadra et al. (16Bhadra S. Maranhao A.C. Ellington A.D. A one-enzyme RT-qPCR assay for SARS-CoV-2, and procedures for reagent production.bioRxiv. 2020; 10.1101/2020.03.29/013342Google Scholar), GT-RTX showed strong performance in RT-qPCR in the presence of SUPERase·In, a DTT-independent commercial RNase A inhibitor mixture. However, when the DTT required for RNaseOUT was added, the ΔRn plateau for the reaction was low (Fig. S3B). Our priority was reliance on components that could be manufactured at GT, and because production of our DTT-dependent GT-rRI was successful (see "RNase inhibitor"), we did not further pursue use of GT-RTX in our master mix. We considered five Taq constructs (Table 2) and two hot-start options. The best yields were obtained when T7-inducible Taq plasmids were transformed and grown in Escherichia coli ArcticExpress with Superior Broth or in E. coli BL21 (DE3) using autoinduction medium or 2xYT broth. Still, expression yields (∼0.5 mg/liter culture at best) were notably less than the other proteins produced as part of this project.Table 2Taq polymerases tested in this projectPlasmid descriptionAntibiotic resistanceExpression E. coli strainpACYCChloramphenicolHB101pAKTaqAmpicillinBL21 (DE3)Nterm His Taq in pET28aKanamycinBL21 (DE3), ArcticExpressCterm His Taq in pET20bAmpicillinBL21 (DE3), ArcticExpressSso7d-Taq in pET20bAmpicillinBL21 (DE3), ArcticExpress Open table in a new tab GT-Taq lacking affinity tags was used in our initial RT-qPCR formulations (see below). Of the purification strategies tested, a short heating step followed by anion-exchange column chromatography, similar to that described by Desai and Pfaffle (19Desai U.J. Pfaffle P.K. Single-step purification of a thermostable DNA polymerase expressed in Escherichia coli.Biotechniques. 1995; 19 (8588916): 780-782PubMed Google Scholar), was the most robust, reproducible, and practical method. To save time and resources, we conducted buffer exchange by using centrifugal devices or a PD-10 column, rather than standard dialysis. The purity of this Taq was less than other preparations we tested (Fig. S4A) (e.g. that published by Engelke et al. (20Engelke D.R. Krikos A. Bruck M.E. Ginsburg D. Purification of Thermus aquaticus DNA polymerase expressed in Escherichia coli.Anal. Biochem. 1990; 191 (2085185): 396-40010.1016/0003-2697(90)90238-5Crossref PubMed Scopus (142) Google Scholar) involving polyethylenimine precipitation and weak cation exchange (not shown)), but this did not negatively impact enzyme performance. GT-Taq was highly active after storage for over 2 months. GT-His-Taq, with a WT sequence and an N-terminal hexahistidine tag and purified only by Ni2+-affinity chromatography (Fig. S4A), was used in the final RT-qPCR formulation (see below). The addition of the hexahistidine tag streamlined protocols by enabling a purification scheme similar to that of GT-MMLV (above) and GT-rRI (below). C-terminally His-tagged Taq polymerase expressed at too low a level to warrant further consideration (not shown). Despite concerns about the possibility of protein contaminants or residual genomic DNA after purification, GT-His-Taq did not appear to benefit from a final anion-exchange step (see "Experimental procedures" and Fig. S4A). If residual genomic DNA is present in our purified polymerase, it apparently does not interfere with probe detection of viral amplicons in RT-qPCR. GT-His-Taq purified in one step was active in PCR (Fig. S4A) and exhibited robust enzyme activity in RT-qPCR (see below) for at least 2 months, after which point the supply was depleted from use in experiments. In addition to GT-His-Taq, we considered the ssod7-Taq chimera, a more efficient Taq polymerase compared with WT Taq (21Wang Y. Prosen D.E. Mei L. Sullivan J.C. Finney M. Vander Horn P.B. A novel strategy to engineer DNA polymerases for enhanced processivity and improved performance in vitro.Nucleic Acids Res. 2004; 32 (14973201): 1197-120710.1093/nar/gkh271Crossref PubMed Scopus (226) Google Scholar). Ssod7-Taq (Fig. S4A) expressed in significantly higher yield (2 mg/liter) than any WT Taq polymerases we tested. Although sso7d-Taq performed as well as WT Taq in PCR and RT-qPCR (not shown), we were unable to identify conditions for storage of this enzyme. This version of Taq shows great promise but would only have full utility once storage issues are resolved. Finally, our attempts to evaluate and develop hot-start technology (22Roux K.H. Optimization and troubleshooting in PCR.Cold Spring Harb. Protoc. 2009; 2009 (pdb.ip66) (20147122)10.1101/pdb.ip66Crossref PubMed Scopus (58) Google Scholar) merit discussion. Hot-start is intended to minimize primer dimer formation and premature extension of PCR products during reaction assembly and reverse transcription (23Chou Q. Russell M. Birch D.E. Raymond J. Bloch W. Prevention of pre-PCR mis-priming and primer dimerization improves low-copy-number amplifications.Nucleic Acids Res. 1992; 20 (1579465): 1717-172310.1093/nar/20.7.1717Crossref PubMed Scopus (609) Google Scholar) by inhibiting Taq polymerase at low temperatures. We evaluated a commercial hot-start antibody (Fig. S4B) alongside two alternative hot-start approaches: Taq mutant I705L (24Davalieva K. Efremov D.G. Substitution of Ile707 for Leu in Klentaq DNA polymerase reduces the amplification capacity of the enzyme.Prilozi. 2009; 30 (20087249): 57-6910.20450/mjcce.2010.173PubMed Google Scholar) and aptamer-based OneTaq® Hot-Start DNA Polymerase (Fig. S4C). Consistent with literature reports, the commercial hot-start antibody and the I705L Taq variant inhibited Taq polymerase at room temperature. However, only the hot-start antibody and, to a lesser extent, aptamer-based OneTaq® Hot-Start DNA Polymerase, inhibited Taq at 37 or 50 °C. Hot-start technologies tested here by RT-qPCR did not noticeably improve threshold cycle (Ct) values or fluorescence signals (data not shown). Although we did not detect RNase contamination in the purified GT enzymes we tested (Fig. S5A), RNase activity is anticipated in human and environmental samples. Mammalian RNase A is inhibited by RI, a leucine-rich repeat protein (25Hofsteenge J. Kieffer B. Matthies R. Hemmings B.A. Stone S.R. Amino acid sequence of the ribonuclease inhibitor from porcine liver reveals the presence of leucine-rich repeats.Biochemistry. 1988; 27 (3219361): 8537-854410.1021/bi00423a006Crossref PubMed Scopus (97) Google Scholar) bearing numerous reduced Cys residues. Inhibition of disulfide bond formation by a reducing agent such as DTT is particularly challenging with RI, because it contains two pairs of adjacent Cys residues (25Hofsteenge J. Kieffer B. Matthies R. Hemmings B.A. Stone S.R. Amino acid sequence of the ribonuclease inhibitor from porcine liver reveals the presence of leucine-rich repeats.Biochemistry. 1988; 27 (3219361): 8537-854410.1021/bi00423a006Crossref PubMed Scopus (97) Google Scholar, 26Kobe B. Deisenhofer J. A structural basis of the interactions between leucine-rich repeats and protein ligands.Nature. 1995; 374 (7877692): 183-18610.1038/374183a0Crossref PubMed Scopus (594) Google Scholar). We focused on porcine RI, which is known to be an effective RNase inhibitor and is amenable to recombinant production (27Klink T.A. Vicentini A.M. Hofsteenge J. Raines R.T. High-level soluble production and characterization of porcine ribonuclease inhibitor.Protein Expr. Purif. 2001; 22 (11437592): 174-17910.1006/prep.2001.1422Crossref PubMed Scopus (30) Google Scholar). Our GT-rRI has an N-terminal His tag because that variant expressed better (1.5 mg/liter) than the C-terminal His tag. However, contrary to prior reports (28Šiurkus J. Neubauer P. Heterologous production of active ribonuclease inhibitor in Escherichia coli by redox state control and chaperonin coexpression.Microb. Cell Fact. 2011; 10 (21824411): 6510.1186/1475-2859-10-65Crossref PubMed Scopus (22) Google Scholar, 29Siurkus J. Neubauer P. Reducing conditions are the key for efficient production of active ribonuclease inhibitor in Escherichia coli.Microb. Cell Fact. 2011; 10 (21554746): 3110.1186/1475-2859-10-31Crossref PubMed Scopus (18) Google Scholar), the presence of DTT in the media did not alter our yield (not shown). Purification was carried out in the presence of fresh DTT. GT-rRI eluted from the Ni2+-affinity column at a high level of purity (Fig. S5B) and did not require further column purification. Inhibition of RNase A by GT-rRI was comparable to commercial RNaseOUT (Fig. S5B). GT-rRI was stored in aliquots of ∼1 mg/ml in the presence of 8 mm DTT and was used in RT-qPCR (see below). No detectable change in inhibitor
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