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Jul 2020

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A One-enzyme RT-qPCR Assay for SARS-CoV-2, and Procedures for Reagent Production
SARS-CoV-2单酶RT-qPCR检测方法及试剂生产程序   

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Abstract

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.

Keywords: Coronavirus (冠状病毒), SARS-CoV-2 (严重急性呼吸综合征冠状病毒2), Quantitative RT-PCR (荧光定量PCR), Reverse transcriptase (反转录酶), TaqMan RT-qPCR (Taqman荧光定量PCR), Nucleic acid diagnostics (核酸诊断)

Background

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).


Herein, we layout protocols for dye-based and TaqMan probe-based RT-qPCR assays for CDC-designed SARS-CoV-2 N gene tests (https://www.fda.gov/media/134922/download). We detail a one-enzyme RTX-based RT-qPCR protocol, as well as a two-enzyme RTX/Taq DNA polymerase-based RT-qPCR protocol. While the dye-based one enzyme and the TaqMan probe-based two enzyme RT-qPCR both exhibit comparable performances in the detection of viral RNA, the TaqMan probe-based assay exhibited lower background. In these, we compare our RTX enzyme preparations, for which we provide a detailed purification protocol, with commercially available reagents.


Our RTX-mediated protocols are as sensitive as those that rely on enzyme combinations or commercial enzymes, and can expedite SARS-CoV-2 tests by reducing the number of kits or key enzymes that must be purchased. These protocols can also form the basis for further assay simplification and democratization via the use of so-called ‘cellular reagents’: polymerase-overexpressing cells that can be directly added to molecular diagnostics assays without loss of sensitivity or specificity (Bhadra et al., 2018).


Materials and Reagents

  1. For RTX polymerase-based RT-qPCR analysis

    1. Barrier tips for pipets

    2. Tris-HCl pH7.5 and 8.0 (Sigma-Aldrich, catalog number: T1503 )

    3. (NH4)2SO4 (Sigma-Aldrich, catalog number: A4413 )

    4. KCl (Sigma-Aldrich, catalog number: P3911 )

    5. MgSO4 (Sigma-Aldrich, catalog number: M7506 )

    6. D-glucose (Sigma-Aldrich, catalog number: 47829 )

    7. 1% Triton® X-100 (New England Biolabs, B9004S ), store at -20 °C

    8. Forward PCR primers (Integrated DNA Technologies, Table 1) (Note a), store at -20 °C, 100 µM Stock

    9. Reverse PCR primers (Integrated DNA Technologies, Table 1) (Note a), store at -20 °C, 100 µM Stock

    10. TaqMan probes (Integrated DNA Technologies, Table 1) (Note a), store at -20 °C

    11. 20x EvaGreen solution (Biotium, #31000), store at 4 °C

    12. 4 mM deoxyribonucleotides (dNTP) (New England Biolabs, N0446S ), store at -20 °C

    13. 5 M Betaine (Sigma-Aldrich, B0300 ), store at 4 °C

    14. 20 U/µl SUPERase·In RNase Inhibitor (Thermo Fisher Scientific, AM2694 ), store at -20 °C

    15. 5 U/µl Taq DNA polymerase (New England Biolabs, M0267L ), store at -20 °C

    16. 0.2 mg/ml RTX polymerase (Note b) (purified by Ellington lab), store at -20 °C

    17. Nuclease-free water (store at room temperature)

    18. qPCR tubes or plates with optical covers(Note c) (Roche, 04729692001 ), store at room temperature

    19. Cold-block or ice

    20. 10x RTX buffer (see Recipes)

    21. 10x ThermoPol buffer (see Recipes)

    Notes:

    1. Individual primer and probe stocks or pre-made primer-probe mixes for the CDC N1, N2, and N3 assays (Table 1) available from Integrated DNA Technologies may be used.

    2. RTX polymerase with proofreading capability (RTX), and RTX polymerase without proofreading capability (RTX Exo-) have been compared.

    3. LightCycler 96 real-time PCR machine and 96-well plates with optical plastic film cover designed for use with LightCycler platform were used for these experiments.


      Table 1. CDC TaqMan RT-qPCR primers and probes for SARS-CoV-2a

      aPrimer and probe sequences adapted from https://www.cdc.gov/coronavirus/2019-ncov/lab/rt-pcr-panel-primer-probes.html; last accessed on May 6th, 2020. According to the CDC, oligonucleotide sequences are subject to future changes as the 2019-Novel Coronavirus evolves. Refer to the CDC website for the latest updates.

      bFAM: 6-carboxyfluorescein; BHQ-1: Black Hole Quencher 1


  2. For purification of RTX DNA polymerase

    1. Sterile culture tubes and flasks

    2. Centrifugation tubes (Nalgene, catalog number: 3115-0050 )

    3. Sterile syringe filter 0.2 µm (PES, VWR, catalog number: 28145-501 )

    4. RTX or RTX Exo- polymerase expression plasmid (Addgene, Plasmid #102787 and #102786 )

    5. Competent E. coli T7 RNA polymerase-based protein expression strain, such as BL21 (DE3) (New England Biolabs, C2527H ), store at -80 °C

    6. Antibiotics such as ampicillin or carbenicillin (Goldbio, catalog number: 4800-94-6 )

    7. Superior BrothTM (Athena Enzyme SystemsTM , catalog number: 0105 ), store at room temperature

    8. 1 M Isopropyl-β-D-thiogalactoside (IPTG) (Sigma Aldrich), store at -20 °C

    9. Ni-NTA agarose (Thermo Fisher Scientific, catalog number: 88223 ), store at 4 °C

    10. Disposable protein purification columns (Thermo Fisher Scientific, catalog number: 29924 )

    11. 20,000 MWCO Dialysis cassette (Thermo Fisher Scientific, catalog number: 66012 )

    12. HiTrapTM Heparin HP column (GE Healthcare, catalog number: 17-0406-01 )

    13. Bis-Tris mini protein gel (Thermo Fisher, catalog number: NP0321BOX )

    14. EDTA-free protease inhibitor tablet (Thermo Scientific, catalog number: A32965 )


    Note: All chemicals were molecular biology or analytical grade and purchased from Sigma-Aldrich (see below for catalog number) unless otherwise indicated.

    1. NaCl (Sigma-Aldrich, catalog number: S7653 )

    2. Imidazole (Sigma-Aldrich, catalog number: I5513 )

    3. Igepal CO-630 (Sigma-Aldrich, catalog number: 542334 )

    4. MgSO4 (Sigma-Aldrich, catalog number: M7506 )

    5. HEW Lysozyme (Sigma-Aldrich, catalog number: L6876 )

    6. Tris (Sigma-Aldrich, catalog number: T1503 )

    7. DTT (Sigma-Aldrich, catalog number: D9779 )

    8. KCl (Sigma-Aldrich, catalog number: P3911 )

    9. Tween-20 (Sigma-Aldrich, catalog number: P9416 )

    10. Purification buffers for RTX DNA polymerase purification, store at 4 °C (see Recipes)

      Resuspension Buffer

      Equilibration Buffer

      Lysis Buffer

      Wash Buffer

      Elution Buffer

      Heparin Buffer A

      Heparin Buffer B

    11. Dialysis buffers for dialysis of RTX DNA polymerase, store at 4 °C (see Recipes)

      Ni-NTA Dialysis Buffer

      Heparin Dialysis Buffer

      Final Dialysis Buffer

Equipment

  1. Magnetic stir bar

  2. Magnetic stirrer

  3. Real-time PCR machine

    Note: LightCycler96 real-time PCR machine from Roche was used for these experiments.

  4. Refrigerated shaker incubator (New Brunswick Scientific, model: Innova44 )

  5. Sonicator (Fisher Scientific, Sonic Dismembrator Model 500 )

  6. Centrifuge (Beckman Coulter, model: Avanti JXN-26 , Fixed angle)

  7. Thermomixer (Eppendorf, model: Thermomixer C )

  8. Fast protein liquid chromatography (FPLC) machine (GE Healthcare, model: AKTA pure )

  9. Fraction collector (GE Healthcare, model: Fraction Collector F9-R )

Software

  1. LightCycler96 software (Roche; available for download from https://lifescience.roche.com/en_us/brands/realtime-pcr-overview.html#software)

Procedure

  1. Dye-based RT-qPCR reaction set up using only RTX DNA polymerase

    1. Set up EvaGreen dye-containing RT-qPCR reactions for CDC SARS-CoV-2 N1, N2, and N3 assays as shown in Table 2. The three assays target three different regions within the nucleocapsid phosphoprotein (N) gene near the 3’-end of the ~30 kb viral genome. In particular, N1 primers (Table 1) amplify a 72 nt long region from position 28287 to 28358 of the genome (GenBank Accession No. MN985325.1). The N2 primers produce a 67 bp amplicon from the genomic position 29164 to 29230. Meanwhile, the N3 primers amplify a 72 nt long region from genomic position 28681 to 28752. RNA isolation should be performed before RT-qPCR.


      Table 2. EvaGreen RT-qPCR reaction mix using only RTX DNA polymerase
      500 " class="layerphoto" src="https://zh-cn.bio-protocol.org/attached/image/20210117/20210117203719_5576.jpg" alt="" />
      aFull length SARS-CoV-2 or MERS-CoV N synthetic RNAs were used in these studies. The RNA copies per reaction (100 to 100,000 copies) are indicated in the relevant figures. Patient saliva is reported to have median SARS-CoV-2 viral loads of 3.3 × 106 copies/ml (To et al., 2020).


    2. Assemble all reaction mixes on a cold-block and directly transfer to a real-time PCR machine programmed to cycle through the steps depicted in Table 3. Include Melt curve analysis steps after qPCR amplification in order to distinguish target amplicons from spurious products.


      Table 3. RTX polymerase-based EvaGreen RT-qPCR cycling conditions
      500 " class="layerphoto" src="https://zh-cn.bio-protocol.org/attached/image/20210117/20210117204025_4262.jpg" alt="" />

      aAssay time may be reduced by shortening the reverse transcription step (step 1) to 15 min and the following denaturation step (step 2) to 2 min.

      bSignal acquisition occurs in the FAM (fluorescein) channel during steps 4 and 7.

      c20 measurements/°C temperature change


  2. RTX polymerase-based TaqMan RT-qPCR reaction set up

    1. In order to perform TaqMan RT-qPCR we developed a one-pot two enzyme system comprising RTX and Taq DNA polymerases. Set up RTX-based TaqMan RT-qPCR reactions for CDC SARS-CoV-2 N1, N2, and N3 assays as shown in Table 4.

    2. Assemble all reaction mixes on a cold-block and directly transfer to a real-time PCR machine programmed to cycle through the steps depicted in Table 5.


      Table 4. RTX polymerase-based TaqMan RT-qPCR reaction mix
      500 " class="layerphoto" src="https://zh-cn.bio-protocol.org/attached/image/20210117/20210117204138_1391.jpg" alt="" />

      aAssay buffer: Duplicate TaqMan RT-qPCR reactions were performed either in 1x ThermoPol buffer or in 1x RTX buffer as indicated to determine optimum buffer conditions.

      bControl reactions lacking RTX polymerase were set up using the same recipe except RTX was replaced with water.


      Table 5. RTX polymerase-based TaqMan RT-qPCR cycling conditions
      500 " class="layerphoto" src="https://zh-cn.bio-protocol.org/attached/image/20210117/20210117204242_0971.jpg" alt="" />
      aAssay time may be reduced by shortening the reverse transcription step (step 1) to 15 min and the following denaturation step (step 2) to 2 min without compromising results.

      bSignal acquisition occurs in the FAM (fluorescein) channel during step 4.


  3. SARS-CoV-2 RT-qPCR using commercial TaqPathTM 1-Step RT-qPCR Master Mix, CG

    1. To perform CDC N1, N2, and N3 SARS-CoV-2 TaqMan RT-qPCR assays using commercial assay master mix, set up reactions using TaqPathTM 1-Step RT-qPCR Master Mix, CG (Thermo Fisher Scientific, Waltham, MA, USA) as shown in Table 6.

    2. Assemble all reactions on a cold-block and then incubate at room temperature (25 °C) for 2 min prior to cycling on a real-time PCR machine using the steps indicated in Table 7.


      Table 6. CDC N1, N2, N3 TaqMan RT-qPCR set up using commercial RT-qPCR master mix
      500 " class="layerphoto" src="https://zh-cn.bio-protocol.org/attached/image/20210117/20210117204209_4242.jpg" alt="" />


      Table 7. TaqPathTM TaqMan RT-qPCR cycling conditions
      500 " class="layerphoto" src="https://zh-cn.bio-protocol.org/attached/image/20210117/20210117204347_4802.jpg" alt="" />

      aSignal acquisition occurs in the FAM (fluorescein) channel during step 4.


  4. Purification of RTX

    1. Transform a T7-based E. coli expression strain, such as BL21(DE3), with a RTX expression plasmid that contains a carboxyl terminal six histidine tag for purification.

    2. On the next day, pick an individual transformant colony and inoculate it into desired sterile media (10.Superior BrothTM) with appropriate antibiotics (e.g., Carbenicillin or Ampicillin 100 µg/ml) and 1-2% glucose Grow this starter culture overnight in shaking incubator at 37 °C.

    3. On day three, inoculate 1 L sterile Superior BrothTM (Athena Enzyme SystemsTM) or other rich media with appropriate antibiotics (e.g., Carbenicillin or Ampicillin 100 µg/ml) in a 4 L Erlenmeyer flask with 250-350 μl of the overnight starter culture. Incubate at 37 °C, 220-250 rpm until the culture reaches OD600 of 0.4-0.7 (0.5-0.6 is ideal).

    4. When the desired OD600 is reached, place flask(s) in 4 °C cold room for 30-45 min and set shaking incubator to 18 °C. Following cold incubation, induce expression of RTX by adding 1 ml of 1 M IPTG to each 1 L of culture. Place flask(s) in the 18 °C shaking incubator for 16-18 h.

    5. Following overnight expression, harvest cells by centrifuging cultures at 4 °C, 5,000 x g for 20 min.

    6. On the ice, resuspend cell pellet in 30 ml cold Lysis Buffer. Transfer the resuspended cell pellet to a small 50 ml beaker with a clean stir bar and place securely in an ice bath. With moderate stirring, sonicate the sample using 40% amplitude and 1 s ON/4 s OFF for 4 min total sonication time.

    7. Centrifuge the resulting lysate at 4 °C, 35,000 x g for 30 min. Carefully transfer the supernatant to a clean ultracentrifugation tube, shake in thermomixer at 400 rpm, 85 °C for 10 min, and then place on ice for 10 min.

    8. Centrifuge the heat-treated lysate at 4 °C, 35,000 x g for 30 min. Carefully transfer the supernatant to a clean tube and filter the clarified lysate using a 0.2 μm filter.

    9. Prepare Ni-NTA agarose columns with a final column volume (CV) of 1 ml using 10 ml PierceTM disposable columns. Equilibrate column with 20 CV Equilibration Buffer.

    10. Apply the clarified lysate from 1 L of expressed culture to a previously equilibrated column and collect the flow-through.

    11. Wash column with 20 CV Equilibration Buffer and collect the flow-through. Wash column with 5 CV Wash Buffer and collect flow-through.

    12. Elute RTX from a column with 5 ml Elution Buffer and transfer to a dialysis cassette with appropriate molecular weight cut-off. Dialyze eluate into 2 L of Ni-NTA Dialysis Buffer for 3-4 hours at 4 °C. Then dialyze eluate into a second 2 L of Ni-NTA Dialysis Buffer overnight at 4 °C.

    13. Pass the dialyzed eluate over an equilibrated 5 ml heparin column (HiTrapTM Heparin HP) and elute along a sodium chloride linear gradient (100 mM to 2 M NaCl) generated from Heparin buffer A and Heparin buffer B mixed by an FPLC machine. The peak corresponding to RTX or its variants could be expected between 40-60% Buffer B.

    14. Collect fraction tubes containing RTX peaks from the fraction collector. Pool the fractions and dialyze into 2 L Heparin Dialysis Buffer for 3-4 h. Then transfer dialysis cassettes into 2 L Final Dialysis Buffer overnight where it is expected that the protein sample volume will decrease significantly. Following the final dialysis, recover the protein sample and determine protein concentration before storing at -20 °C. The RTX stock is typically kept concentrated (5-10 mg/ml) in the Final Dialysis Buffer. If a lower concentration is required (e.g., 0.2 mg/ml working solution), Final Dialysis Buffer was used to dilute the RTX stock to the desired final concentration. An example of RTX purification is shown in Figure 7.

    15. A similar protocol should work for Taq and other thermostable DNA polymerases, but may require different dialysis and storage buffers.

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.


Example data for RTX polymerase-based TaqMan RT-qPCR

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

  1. 10x RTX buffer

    600 mM Tris-HCl

    250 mM (NH4)2SO4

    100 mM KCl

    20 mM MgSO4

    pH 8.4, 25 °C

  2. 10x ThermoPol buffer

    200 mM Tris-HCl

    100 mM (NH4)2SO4

    100 mM KCl

    20 mM MgSO4

    1% Triton® X-100

    pH 8.8, 25 °C


Buffers for RTX DNA polymerase purification

  1. Resuspension Buffer

    50 mM Phosphate Buffer, pH 7.5

    300 mM NaCl

    20 mM imidazole

    0.1% Igepal CO-630 (non-toxic Non-idet P40 equivalent)

    5 mM MgSO4

  2. Equilibration Buffer

    50 mM Phosphate Buffer, pH 7.5

    300 mM NaCl 20 mM imidazole

  3. Lysis Buffer

    30 ml Resuspension Buffer

    1x EDTA-free protease inhibitor tablet

    30-60 mg HEW Lysozyme

  4. Wash Buffer

    50 mM Phosphate Buffer, pH 7.5

    300 mM NaCl

    50 mM imidazole

  5. Elution Buffer

    50 mM Phosphate Buffer, pH 7.5

    300 mM NaCl

    250 mM imidazole

  6. Heparin Buffer A

    40 mM Tris-HCl, pH 7.5

    100 mM NaCl

    0.1% Igepal CO-630 (non-toxic Non-idet P40 equivalent)

  7. Heparin Buffer B

    40 mM Tris-HCl, pH 7.5

    2 M NaCl

    0.1% Igepal CO-630 (non-toxic Non-idet P40 equivalent)


Buffers for dialysis of RTX DNA polymerase

  1. Ni-NTA Dialysis Buffer (2 L x2)

    40 mM Tris-HCl, pH 7.5

    100 mM NaCl

    10 mM beta-mercapto ethanol (BME) or 1 mM 1,4-Dithiothreitol (DTT)

    0.1% Igepal CO-630 (non-toxic Non-idet P40 equivalent)

  2. Heparin Dialysis Buffer (2 L)

    50 mM Tris-HCl, pH 8.0

    50 mM KCl

    0.1% Tween-20

    0.1% Igepal CO-630 (non-toxic Non-idet P40 equivalent)

  3. Final Dialysis Buffer (1 L)

    50% Glycerol

    50 mM Tris-HCl, pH 8.0

    50 mM KCl

    0.1% Tween-20

    0.1% Igepal CO-630 (non-toxic Non-idet P40 equivalent)

    1 mM DTT

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.

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  9. To, K. K., Tsang, O. T., Yip, C. C., Chan, K. H., Wu, T. C., Chan, J. M., Leung, W. S., Chik, T. S., Choi, C. Y., Kandamby, D. H., Lung, D. C., Tam, A. R., Poon, R. W., Fung, A. Y., Hung, I. F., Cheng, V. C., Chan, J. F. and Yuen, K. Y. (2020). Consistent Detection of 2019 Novel Coronavirus in Saliva. Clin Infect Dis 71(15): 841-843.

简介

[摘要]鉴于持续进行的COVID-19大流行的规模,可靠,可扩展的测试需求以及试剂短缺的可能性(尤其是在资源匮乏的环境中),我们开发了一种n RT-qPCR分析方法,该方法依赖于其他方法与常规病毒逆转录酶相比,热稳定的逆转录酶/ DNA聚合酶(RTX)(Ellefson等,2016)。在这里,我们显示RTX与CDC认可并以试剂盒形式验证的其他检测方法具有可比性。我们演示了两种RTX使用模式-(i)仅需要RTX聚合酶的基于染料的RT-qPCR分析,以及(ii)使用RTX和Taq DNA聚合酶组合的TaqMan RT-qPCR分析(因为RTX核酸外切酶不降级ea TaqMan探针)。我们还提供了纯化该替代试剂RTX的简单方法。我们预计,在资源匮乏或需要的地方,研究人员可以获取可用的构建体,并开始开发自己的测定方法,而不论它们存在的调控框架如何。


[背景]尽管已采用多种病毒检测方法来检测SARS-CoV-2感染,包括各种分子诊断和免疫诊断测试,但逆转录酶定量聚合酶链反应(RT-qPCR)仍然是主要且最敏感的方法SARS-CoV-2检测试验(D'Cruz等,2020 ; Tang等,2020 )。RT-qPCR的首要地位在很大程度上是因为基于抗体的测试以及快速核酸诊断平台(如Abbott IDNow)通常灵敏度低下,尤其是在早期感染期间患者的病毒载量普遍较低(Basu等等人,2020;D'Cruz等人,2020)。鉴于在含COVID-19爆发的早期诊断中的重要性(派克,2020) ,需要一个快速和可升级的RT-qPCR的设置是迫在眉睫。不幸的是,规模化基于RT-qPCR的测试所必需的各种试剂的短缺日益增加(Pettit等,2020)。的PCR平台的主要制造商无法向上扩展的生产足够数量的测试,以供应资源贫乏。甚至在资源贫乏的环境中尝试开发自己的解决方案时,研究人员,临床医生和公共卫生官员通常也缺乏必要的试剂(包括酶)来开发测试程序(Kavanagh等人,2020年)。

在此,我们布局用于染料和基于探针的TaqMan RT-qPCR测定为CDC设计的SARS-CoV的-2 N基因测试(协议https://www.fda.gov/media/134922/download)。我们详细介绍了一种基于酶RTX的RT-qPCR协议,以及两种基于酶RTX / Taq DNA聚合酶的RT-qPCR协议。尽管基于染料的一种酶和基于TaqMan探针的两种酶RT-qPCR在检测病毒RNA方面均表现出可比的性能,但基于TaqMan探针的分析却具有较低的背景。在这些中,我们将我们提供详细的纯化方案的RTX酶制剂与市售试剂进行比较。

我们的RTX介导的方案与依赖酶组合或商业酶的方案一样敏感,并且可以通过减少必须购买的试剂盒或关键酶的数量来加快SARS-CoV-2测试。这些协议还可通过使用所谓的“细胞试剂”为进一步简化分析和实现民主化奠定基础:过表达聚合酶的细胞可直接添加到分子诊断分析中,而不会损失灵敏度或特异性(Bhadra等,2018)。

关键字:冠状病毒, 严重急性呼吸综合征冠状病毒2, 荧光定量PCR, 反转录酶, Taqman荧光定量PCR, 核酸诊断

材料和试剂
用于基于RTX聚合酶的RT-qPCR分析
1.移液器的壁垒提示     
2. Tris-HCl pH7.5和8.0(Sigma-Aldrich,目录号:T1503)     
3. (NH 4 )2 SO 4 (Sigma-Aldrich,目录号:A4413)     
4. KCl(西格玛奥德里奇,目录号:P3911)     
5. MgSO 4 (西格玛奥德里奇,目录号:M7506)     
6. D-葡萄糖(西格玛奥德里奇,目录号:47829)     
7. 1%的Triton ® X-100(新英格兰生物实验室,B9004S) ,储存在-20℃下     
8.正向PCR引物(集成的DN A技术,表1 )(注a),储存在-20°C,100 µM     
9.反向PCR引物(DNA集成技术,表1 )(注a),储存在-20°C,100 µM     
10. TaqMan探针(DNA集成技术,表1 )(注a),储存在-20°C 
11. 20x EvaGreen溶液(Biotium,#31000),在4°C下储存 
12. 4 mM脱氧核糖核苷酸(dNTP)(New England Biolabs,N0446S),储存在-20°C 
13. 5 M甜菜碱(Sigma-Aldrich,B0300),储存在4°C 
14. 20 U / µl SUPERase·In RNase抑制剂(Thermo Fisher Scientific,AM2694),储存在-20°C 
15. 5 U / µl Taq DNA聚合酶(New England Biolabs,M0267L),储存在-20°C 
16. 0.2 mg / ml RTX聚合酶(注b)(由Ellington实验室纯化),储存在-20°C 
17.无核酸酶的水(室温保存) 
18.带光学盖的qPCR试管或板(注c)(Roche ,04729692001),在室温下保存 
19.冷块或冰 
20. 10 x RTX缓冲区(请参阅食谱) 
21. 10 x ThermoPol缓冲液(请参阅食谱) 
注意事项:

个别引物和探针的股票或为CDC N1,N2预先制作的引物-探针混合物,和N3测定(表1 ,可以使用),得自集成DNA技术。
比较了具有校对能力的RTX聚合酶(RTX)和不具有校对能力的RTX聚合酶(RTX Exo-)。
这些实验使用了LightCycler 96实时PCR机和设计用于LightCycler平台的带有光学塑料薄膜盖的96孔板。

表1 。CDC TaqMan RT-qPCR的SARS-CoV-2 a引物和探针

名称

描述

顺序b

2019-nCoV_N1-F

2019-nCoV_N1正向引物

5'-GAC CCC AAA ATC AGC GAA AT-3'

2019-nCoV_N1-R

2019-nCoV_N1反向引物

5'-TCT GGT TAC TGC CAG TTG AAT CTG-3'

2019-nCoV_N1-P

2019-nCoV_N1探针

5'-FAM-ACC CCG CAT TAC GTT TGG TGG ACC-BHQ1-3'

2019-nCoV_N2-F

2019-nCoV_N2正向底漆

5'-TTA CAA ACA TTG GCC GCA AA-3'

2019-nCoV_N2-R

2019-nCoV_N2反向底漆

5'-GCG CGA CAT TCC GAA GAA-3'

2019-nCoV_N2-P

2019-nCoV_N2探针

5'-FAM-ACA ATT TGC CCC CAG CGC TTC AG-BHQ1-3'

2019-nCoV_N3-F

2019-nCoV_N3正向引物

5'-GGG AGC CTT GAA TAC ACC AAA A-3'

2019-nCoV_N3-R

2019-nCoV_N3反向底漆

5'-TGT AGC ACG ATT GCA GCA TTG-3'

2019-nCoV_N3-P

2019-nCoV_N3探针

5'-FAM-AYC ACA TTG GCA CCC GCA ATC CTG-BHQ1-3'

从https://www.cdc.gov/coronavirus/2019-ncov/lab/rt-pcr-panel-primer-probes.ht ml改编的引物和探针序列; 上一次访问时间是2020年5月6日。根据CDC,随着2019年新颖的冠状病毒的发展,寡核苷酸序列可能会发生未来的变化。请访问CDC网站以获取最新更新。

b FAM:6-羧基荧光素;BHQ-1:黑洞熄灭器1


用于纯化RTX DNA聚合酶
1.无菌培养管和烧瓶     
2.离心管(Nalgene,目录号:3115-0050)     
3.无菌注射器过滤器0.2 µm(PES,VWR,目录号:28145-501)     
4. RTX或RTX外切聚合酶表达质粒(Addgene,质粒#102787和#102786)     
5.主管大肠杆菌T7 RNA聚合酶为基础的蛋白表达菌株,如BL21(DE3)(New England Biolabs公司,C2527H),储存在-80℃下     
6.抗生素,例如氨苄青霉素或羧苄青霉素(Goldbio,目录号:4800-94-6)     
7. Superior Broth TM (雅典娜酶系统TM ,目录号:0105),在室温下保存     
8. 1 M异丙基-β-D-硫代半乳糖苷(IPTG)(Sigma Aldrich),储存在-20°C     
9. Ni-NTA琼脂糖(Thermo Fisher Scientific,目录号:88223),在4°C下储存     
10.一次性蛋白质纯化柱(Thermo Fisher Scientific,目录号:29924) 
11. 20,000 MWCO透析盒(Thermo Fisher Scientific,目录号:66012) 
12. HiTrap TM肝素HP色谱柱(GE Healthcare,目录号:17-0406-01) 
13.的Bis-Tris迷你蛋白凝胶(热费舍尔,Ç atalog号:NP0321BOX) 
14.无EDTA的蛋白酶抑制剂片剂(Thermo Scientific ,目录号:A32965) 

注意:除非另有说明,否则所有化学药品均为分子生物学或分析纯,购自Sigma-Aldrich(目录号见下文)。

15. NaCl(西格玛奥德里奇,目录号:S7653) 
16.咪唑(Sigma-Aldrich,目录号:I5513) 
17. Igepal CO-630(Sigma-Aldrich,目录号:542334) 
18. MgSO 4 (Sigma-Aldrich,目录号:M7506) 
19. HEW溶菌酶(Sigma-Aldrich,目录号:L6876) 
20. Tris(Sigma-Aldrich,目录号:T1503) 
21. DTT(西格玛奥德里奇,目录号:D9779) 
22. KCl(西格玛奥德里奇,目录号:P3911) 
23. Tween-20(Sigma-Aldrich,目录号:P9416) 
24.用于RTX DNA聚合酶纯化的纯化缓冲液,储存在4°C下(请参阅食谱) 
重悬缓冲液

平衡缓冲液

裂解缓冲液

洗涤缓冲液

洗脱缓冲液

肝素缓冲液A

肝素缓冲液B

25.用于RTX DNA聚合酶透析的透析缓冲液,储存在4°C下(请参阅食谱) 
Ni-NTA透析缓冲液

肝素透析缓冲液

最终透析缓冲液


设备


磁力搅拌棒
电磁搅拌机
实时PCR机
注意:这些实验使用了罗氏公司的LightCycler96实时PCR机。

冷冻摇床培养箱(New Brunswick Scientific,型号:Innova44)
声波发生器(Fisher Scientific,500型声波分解器)
离心机(贝克曼库尔特(Beckman Coulter),型号:Avanti JXN-26,固定角度)
Thermomixer(Eppendorf ,型号:Thermomixer C)
快速蛋白质液相色谱(FPLC)机(GE Healthcare ,型号:AKTA pure)
级分收集器(GE Healthcare公司,型号:˚F raction集电极F9-R)

软件


LightCycler96软件(Roche;可从https://lifescience.roche.com/en_us/brands/realtime-pcr-overview.ht ml #software下载)

程序


仅使用RTX DNA聚合酶建立基于染料的RT-qPCR反应
设置CDC SARS-CoV-2 N1,N2和N3分析的EvaGreen染料染色RT-qPCR反应,如表2所示。这三种测定法针对的是〜30 kb病毒基因组3'末端附近的核衣壳磷蛋白(N)基因内的三个不同区域。特别地,N1引物(表1 )从基因组(GenBank登录号MN985325.1)的位置28287到28358扩增72nt长的区域。N2引物从基因组位置29164至29230产生67bp的扩增子。同时,N3引物从基因组位置28681至28752扩增72nt长的区域。RNA分离应在RT-qPCR之前进行。

表2 。仅使用RTX DNA聚合酶的EvaGreen RT-qPCR反应混合物


体积(微升)

最终浓度

RNA样品一

5


5 µM正向PCR引物

2.5

500 nM

5 µM反向PCR引物

2.5

500 nM

20 x EvaGreen

1.25

1个

10 x RTX缓冲区

2.5

1个

4毫米dNTP

2.5

400微米

20 U / µl SUPERase·In RNase抑制剂

1个

0.8 U /微升

RTX或RTX Exo- 0.2 mg / ml

0.5

4 ng /微升

5M甜菜碱

2.5

50万



4.75


总反应量

25


一个全长SARS-CoV的-2或MERS-CoV的ñ合成的RNA在这些研究中使用。每个反应的RNA拷贝数(100到100,000个拷贝)在相关图中显示。据报道,患者唾液中的SARS-CoV-2病毒载量中位数为3.3×10 6拷贝/ ml (To等人,2020年)。


将所有反应混合物组装在冷块上,然后直接转移至实时PCR仪中,该PCR仪经过编程可循环执行表3中所示的步骤。已包含在顺序的qPCR扩增后UDE熔体曲线分析步骤,从杂散产物区分靶扩增子。
表3 。基于RTX聚合酶的EvaGreen RT-qPCR循环条件



温度

持续时间

信号采集

1个

60°摄氏度

30分钟

没有

2个

95°C

10分钟

没有

步骤3和4的55个循环

3

95°C

15秒

没有

4 b

55°摄氏度

30秒



步骤5、6和7中的融解曲线分析

5

95°C

10秒

没有

6

65°摄氏度

60秒

没有

7

97°摄氏度

1秒

连续c

一测定时间可通过缩短被减小的反转录步骤(小号TEP 1)至15分钟和下面的变性步骤(小号TEP 2)至2分钟。

b期间信号采集发生在FAM(荧光素)信道小号TEPS 4和7。

c 20测量/°C温度变化


基于RTX聚合酶的TaqMan RT-qPCR反应设置
为了进行TaqMan RT-qPCR,我们开发了一种包含RTX和Taq DNA聚合酶的一锅两用酶系统。设置为CDC SARS-CoV的-2 N1,N2和N3的测定基于RTX-的TaqMan RT-qPCR反应中所示的Ta BLE 4 。
在冷块上组装所有反应混合物,然后直接转移到实时PCR仪中,该PCR仪经过编程可循环执行表5中所示的步骤。

表4 。基于RTX聚合酶的TaqMan RT-qPCR反应混合物


体积(微升)

最终浓度

RNA样本

5


6.7 µM正向PCR引物

1.5

402 nM

6.7 µM反向PCR引物

1.5

402 nM

1.7 µM TaqMan探针

1.5

102 nM

10 X测定缓冲液一

2.5

1个

4毫米dNTP

2.5

400微米

20 U / µl SUPERase·In RNase抑制剂

1个

0.8 U /微升

5 U / µl Taq DNA聚合酶

0.5

0.1 U /微升

RTX或RTX Exo- 0.2 mg / ml或水b

0.5

4 ng /微升

5M甜菜碱

2.5

50万



6


总反应量

25


a检测缓冲液:按照指示在1 x ThermoPol缓冲液或1 x RTX缓冲液中进行重复的TaqMan RT-qPCR反应,以确定最佳缓冲液条件。

b使用相同的配方设置缺少RTX聚合酶的对照反应,只是用水代替RTX。


表5 。基于RTX聚合酶的TaqMan RT-qPCR循环条件

步骤1一

60°摄氏度

30分钟

步骤2 a

95°C

10分钟

步骤3和4的55个循环

第三步

95°C

15秒

步骤4 b

55°摄氏度

30秒

在不影响结果的前提下,可通过将反转录步骤(s tep 1)缩短至15分钟并将随后的变性步骤(s tep 2)缩短至2 min来缩短测定时间。

b在步骤4中,信号采集发生在FAM(荧光素)通道中。


使用市售TaqPath TM 1-Step RT-qPCR预混液的SARS-CoV-2 RT-qPCR
要使用商品化的预混液进行CDC N1,N2和N3 SARS-CoV-2 TaqMan RT-qPCR检测,请使用CG TaqPath TM 1-Step RT-qPCR预混液(Thermo Fisher Scientific,Waltham,MA,美国)如表6所示。
装配上所有反应一冷-块然后在室温下孵育循环上的实时使用步骤PCR机中指示(25℃)2分钟之前和表7 。

表6 。使用商用RT-qPCR预混液建立CDC N1,N2,N3 TaqMan RT-qPCR


体积(微升)

RNA样本

5

6.7 µM正向PCR引物

1.5

6.7 µM反向PCR引物

1.5

1.7 µM TaqMan探针

1.5

4 x TaqPath TM预混液

5



5.5

总反应量

20


TABL ê 7 。TaqPath TM TaqMan RT-qPCR循环条件

第1步

50°摄氏度

15分钟

第2步

95°C

2分钟

步骤3和4的45个循环

第三步

95°C

3秒

步骤4

55°摄氏度

30秒一

一个信号采集在FAM(荧光素)信道的步骤4期间发生。



RTX的纯化
用包含羧基末端六个组氨酸标签的RTX表达质粒转化基于T7的大肠杆菌表达菌株,例如BL21(DE3),以进行纯化。 
第二天,挑个体转化体的菌落,接种它成所需的无菌培养基(10高级肉汤TM )用适当的抗生素(例如,羧苄青霉素或氨苄青霉素100微克/米升)和1-2%的葡萄糖成长此起始培养过夜在37°C的振荡培养箱中。             
在第三天,接种1升无菌高级肉汤TM (雅典娜化酶系统TM )或用适当的抗生素(其他富媒体例如,羧苄青霉素或氨苄青霉素100微克/米升在4L锥形烧瓶中)与250-350μ升的过夜发酵剂文化。在37°C,220-250 rpm下孵育,直到培养物的OD 600为0.4-0.7(理想的是0.5-0.6)。              
当在所希望的OD 600达到,代替烧瓶(一个或多个)在4℃冷室中静置30-45分钟和组摇动孵化器至18℃。冷培养后,通过向每1 L培养物中添加1 ml 1 M IPTG诱导RTX表达。将烧瓶放在18°C的振荡培养箱中16-18小时。
过夜表达后,通过在4°C ,5、000 xg下离心20分钟培养物来收获细胞。
上所述在30冰,悬浮细胞沉淀毫升冷裂解缓冲液。传输重新悬浮细胞沉淀至小50 ml的烧杯用一个在冰浴中清洁的搅拌棒和地方牢固。在适度搅拌下,使用40%振幅和1 s ON / 4 s OFF对样品进行超声处理,总超声处理时间为4分钟。
在4°C,35,000 xg下将所得的裂解物离心30分钟。小心地将上清液转移至干净的超速离心管中,在温度为85℃,400 rpm的热混合器中摇动10分钟,然后在冰上放置10分钟。 
将热处理过的裂解物在4°C,35,000 xg下离心30分钟。小心地将上清液转移到干净的试管中,并使用0.2μm过滤器过滤澄清的裂解液。
制备Ni-NTA琼脂糖柱用一个的最后1个柱体积(CV)毫升,用10个毫升皮尔斯TM一次性列。用20 CV平衡缓冲液平衡色谱柱。
将来自1 L表达培养物的澄清裂解物应用于先前平衡的色谱柱,并收集流通量。 
用20 CV平衡缓冲液洗涤色谱柱,并收集流通量。用5 CV洗涤缓冲液洗涤色谱柱,并收集流通量。 
洗脱RTX从一个用5柱毫升洗脱缓冲液并转移到具有合适的分子量截断透析盒。在4°C下将洗脱液透析至2 L Ni-NTA透析缓冲液中3-4小时。然后在4°C过夜,将洗脱液渗入第二个2 L的Ni-NTA透析缓冲液中。
将透析的洗脱液通过平衡的5 ml肝素柱(HiTrap TM Heparin HP),然后沿由FPLC机器混合的肝素缓冲液A和肝素缓冲液B生成的氯化钠线性梯度(100 mM至2 M NaCl)洗脱。可以预期在40-60%Buffer B之间对应于RTX或其变体的峰。 
从馏分收集器收集含有RTX峰的馏分管。合并级分并在2 L肝素透析缓冲液中透析3-4小时。然后将透析盒转移到2 L最终透析缓冲液中过夜,以期预期蛋白质样品量会大大减少。在最终直径裂解,回收的蛋白质样品并在-20℃下储存之前确定蛋白质浓度。RTX储备液通常在最终透析缓冲液中保持浓缩(5-10 mg / ml )。如果需要较低的浓度(例如0.2 mg / ml的工作溶液),则使用最终透析缓冲液将RTX储备液稀释至所需的最终浓度。RTX纯化的一个例子如图7所示。
类似的方案应适用于Taq和其他热稳定的DNA聚合酶,但可能需要不同的透析和储存缓冲液。 

数据分析


使用LightCycler96软件分析RT-qPCR数据。


基于染料的仅RTX的RT-qPCR的示例数据

图1和2显示了使用合成RNA模板进行的SARS-CoV-2 RT-qPCR测试的代表性结果,仅RTX或RTX Exo-DNA聚合酶。这些结果表明,单独的RTX DNA聚合酶具有或不具有校对能力,可以支持基于染料的RT-qPCR分析。在我们手中,全长RTX DNA聚合酶比Exo-版本稍好,尤其是在N3分析中。





图1.使用合成RNA和RTX DNA聚合酶的指定副本进行的CDC SARS-CoV-2 N1,N2和N3 RT-qPCR分析。小图A,B和C描述了使用EvaGreen染料实时测量的N1,N2和N3分析。描绘了来自包含SARS-CoV-2合成N RNA的100,000(黑色迹线),10,000(蓝色迹线),1,000(红色迹线)和100(粉红色迹线)拷贝的反应的扩增曲线。阴性对照反应不含模板(灰色痕迹)或包含100,000份MERS-CoV合成N RNA的拷贝(绿色痕迹)。图D,E和F描绘了使用LightCycler96软件中的“ Tm调用”分析确定的扩增子的熔解峰。注意,在阴性对照中观察到的扩增子是伪造的,与目标衍生的扩增子相比,其独特的解链温度表明。将所有测定的平均Cq值制成表格。





图2. CDC SARS-CoV-2 N1,N2和N3 RT-qPCR分析法使用指定的合成RNA和RTX Exo-DNA聚合酶进行。小图A,B和C描述了使用EvaGreen染料实时测量的N1,N2和N3分析。描绘了来自包含SARS-CoV-2合成N RNA的100,000(黑色迹线),10,000(蓝色迹线),1,000(红色迹线)和100(粉红色迹线)拷贝的反应的扩增曲线。阴性对照反应不含模板(灰色痕迹)或包含100,000份MERS-CoV合成N RNA的拷贝(绿色痕迹)。图D,E和F描绘了使用LightCycler96软件中的“ Tm调用”分析确定的扩增子的熔解峰。将所有测定的平均Cq值制成表格。


基于RTX聚合酶的TaqMan RT-qPCR的示例数据

使用合成RNA模板进行的基于RTX聚合酶的SARS-CoV-2 TaqMan RT-qPCR测试的代表性结果如图3和图4所示。当与Taq DNA聚合酶结合使用时,RTX和RTX Exo-都能够支持所有三种CDC SARS-CoV-2分析的一锅TaqMan RT-qPCR分析。在两种测试缓冲液条件下(1 x ThermoPol与1 x RTX缓冲液),与RTX Exo-聚合酶相比,RTX聚合酶在所有三种CDC分析中均产生更一致的扩增曲线。





图3. CDC SARS-CoV-2 N1,N2和N3 TaqMan RT-qPCR分析法在1X ThermoPol缓冲液中进行,使用指定的合成RNA副本以及RTX或RTX Exo-和Taq DNA聚合酶进行。小图A,B和C描述了同时包含RTX和Taq DNA聚合酶的TaqMan分析。D,E和F组描绘了仅包含Taq DNA聚合酶的TaqMan分析。G,H和I组描绘了包含RTX Exo-和Taq DNA聚合酶的TaqMan分析。描绘了来自包含SARS-CoV-2合成N RNA的100,000(黑色迹线),10,000(蓝色迹线),1,000(红色迹线)和100(粉红色迹线)拷贝的反应的扩增曲线。阴性对照反应不包含模板(灰色痕迹),或者包含100,000份来自MERS-CoV的合成N RNA(绿色痕迹)。将所有测定的Cq值制成表格。





图4. CDC SARS-CoV-2 N1,N2和N3 TaqMan RT-qPCR分析法在1X RTX缓冲液中进行,使用指定的合成RNA副本以及RTX或RTX Exo-和Taq DNA聚合酶进行。小图A,B和C描述了包含RTX和Taq DNA聚合酶的TaqMan分析。D,E和F组描绘了包含RTX Exo-和Taq DNA聚合酶的TaqMan分析。描绘了来自包含SARS-CoV-2合成N RNA的100,000(黑色迹线),10,000(蓝色迹线),1,000(红色迹线)和100(粉红色迹线)拷贝的反应的扩增曲线。阴性对照反应不含模板(灰色痕迹)或包含100,000份MERS-CoV合成N RNA的拷贝(绿色痕迹)。将所有测定的Cq值制成表格。


TaqPath TM TaqMan RT-qPCR的示例数据

图5描述了CDC N1,N2和N3 TaqMan qRT-PCR反应的典型结果,这些反应是使用合成RNA和TaqPath TM商业RT-qPCR预混液进行的。





图5. CDC SARS-CoV-2 N1,N2和N3 TaqMan RT-qPCR测定法,使用指定的合成RNA副本和TaqPath TM商业RT-qPCR预混液进行。描绘了来自包含SARS-CoV-2合成N RNA的100,000(黑色迹线),10,000(蓝色迹线),1,000(红色迹线)和100(粉红色迹线)拷贝的反应的扩增曲线。阴性对照反应不含模板(灰色痕迹)或包含100,000份MERS-CoV合成N RNA的拷贝(绿色痕迹)。将所有测定的Cq值制成表格。


病毒RT(在TaqPath TM商业预混液中)或RTX的结果在图6中得到了概括。可以看出,基于RTX的TaqMan分析与金标准TaqPath分析具有相当的灵敏度和特异性。尽管在ThermoPol缓冲液中进行的所有三个基于RTX的TaqMan测定始终产生Cq值,但与通过商业TaqPath TM Master Mix获得的Cq值相比,这些值有些延迟。相反,基于RTX的TaqMan分析在RTX缓冲液中执行时,产生的Cq值更接近于使用商业预混液获得的Cq值。





图6.基于RTX和基于TaqPath的SARS-CoV-2 TaqMan RT-qPCR分析的Cq值比较





图7.通过SDS-PAGE分析RTX纯化的一个例子。在4%至12%的Bis-Tris mini蛋白凝胶上的NuPAGE TM上开发了总共十至四十微克的纯化RTX 。RTX带出现在100kDa大小标记附近。1个ST泳道:10微克,2次车道:20微克,3次泳道:30微克,4个泳道:40微克。


笔记


总体而言,RTX既可以用作嵌入染料(例如EvaGreen)的RT-qPCR的单一酶,也可以作为基于TaqMan的测定的RT成分。在最低的RNA浓度(100拷贝)下,金标准TaqPath分析通常会在Cq值为ca时发出信号。32.更健壮的全长RTX本身实际上表现更好,Cq值通常为29-30(尽管N3引物组提供了更高的信号和更高的背景)。当用作病毒RT的替代品时,RTX总体上敏感性略低,Cq值通常在32-35之间。

通过获得RTX表达载体并纯化热稳定的逆转录酶,应该证明可以进行RT-qPCR反应,其灵敏度与已批准试剂盒中观察到的灵敏度相似,几乎没有假阳性结果。可从Addgene(pET_RTX:https : //www.addgene.org/102787/和pET_RTX_(exo-):https ://www.addgene.org/获得有关6xHis标记的RTX和RTX Exo蛋白表达的质粒和序列信息)102786 /),也可以通过https://reclone.org/获得。


菜谱


1. 10 x RTX缓冲区     
600毫米Tris-HCl

250毫米(NH 4 )2 SO 4

100毫米氯化钾

20毫米MgSO 4

pH值8.4 ,25℃下

2. 10 x ThermoPol缓冲液     
200毫米Tris-HCl

100毫米(NH 4 )2 SO 4

100毫米氯化钾

20毫米MgSO 4

1%的Triton ® X-100

pH值8.8 ,25℃下


RTX DNA聚合酶纯化缓冲液

3.重悬缓冲液     
50 mM磷酸盐缓冲液,pH 7.5

300毫米氯化钠

20 mM咪唑

0.1%Igepal CO-630(等效于无毒,非理想P40)

5毫米MgSO 4

4.平衡缓冲液     
50 mM磷酸盐缓冲液,pH 7.5

300 mM氯化钠20 mM咪唑

5.裂解缓冲液     
30 ml重悬浮缓冲液

1x不含EDTA的蛋白酶抑制剂片剂

30-60 mg HEW溶菌酶

6.洗涤缓冲液     
50 mM磷酸盐缓冲液,pH 7.5

300毫米氯化钠

50 mM咪唑

7.洗脱缓冲液     
50 mM磷酸盐缓冲液,pH 7.5

300毫米氯化钠

250 mM咪唑

8.肝素缓冲液A     
40 mM Tris-HCl,pH 7.5

100毫米氯化钠

0.1%Igepal CO-630(等效于无毒,非理想P40)

9.肝素缓冲液B     
40 mM Tris-HCl,pH 7.5

2 M氯化钠

0.1%Igepal CO-630(等效于无毒,非理想P40)


RTX DNA聚合酶透析缓冲液

10. Ni-NTA透析缓冲液(2 L x2 ) 
40 mM Tris-HCl,pH 7.5

100毫米氯化钠

10 mMβ-巯基乙醇(BME)或1 mM 1,4-二硫苏糖醇(DTT)

0.1%Igepal CO-630(等效于无毒,非理想P40)

11.肝素透析缓冲液(2 L) 
50 mM Tris-HCl,pH 8.0

50毫米氯化钾

0.1%吐温20

0.1%Igepal CO-630(等效于无毒,非理想P40)

12.最终透析缓冲液(1升) 
50%甘油

50 mM Tris-HCl,pH 8.0

50毫米氯化钾

0.1%吐温20

0.1%Igepal CO-630(等效于无毒,非理想P40)

1毫米DTT


致谢


我们感谢德克萨斯大学西南医学中心Schoggins实验室在使合成RNA迅速用于这些测定的过程中做出的杰出贡献。我们要感谢Promega公司(UTA18-000656),美国国立卫生研究院(1R01EB027202-01A1)和韦尔奇基金会(F-1654)的资助。


利益争夺


德克萨斯大学董事会已向Promega Corporation授予涉及RTX的IP许可。


参考文献


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引用: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.
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Inyup Paik
University of Texas at Austin Austin作者
Dear Readers,

Please find His tag versions of RTX expression plasmids at Addgene ID 145028 and 145029. Please find these plasmids at https://www.addgene.org/Andrew_Ellington/
2021/3/26 8:10:29 回复