Comparison of Commercially Available Thermostable DNA Polymerases with Reverse Transcriptase Activity in Coupled Reverse Transcription Polymerase Chain Reaction Assays

TL;DR

This paper compares artificial DNA polymerases with reverse transcriptase activity to traditional enzyme mixtures in RT-PCR assays for detecting RNA, including SARS-CoV-2.

Contribution

The study evaluates the performance of artificial thermostable DNA polymerases with reverse transcriptase activity in RT-PCR compared to conventional enzyme mixtures.

Findings

Artificial DNA polymerases are suitable for SARS-CoV-2 RNA detection in RT-PCR.

Artificial enzymes are less effective for long-fragment RT-PCR amplification.

Both end-point and real-time RT-PCR assays were used to compare enzyme performance.

Abstract

Reverse transcription polymerase chain reaction (RT-PCR) is an important tool for the detection of target RNA molecules and the assay of RNA pathogens. Coupled RT-PCR is performed with an enzyme mixture containing a reverse transcriptase and a thermostable DNA polymerase. To date, several biotechnological companies offer artificial thermostable DNA polymerases with a built-in reverse transcriptase activity for use in the coupled RT-PCR instead of the enzyme mixtures. Here, we compared the artificial DNA polymerases and conventional enzyme mixtures for the RT-PCR by performing end-point and real-time RT-PCR assays using severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV2) RNA and endogenous mRNA molecules as templates. We found that the artificial enzymes were suitable for different RT-PCR applications, including SARS-CoV2 RNA detection but not for long-fragment RT-PCR amplification.

Figures

Full text

1. Introduction

The coronavirus disease 2019 (COVID-19) pandemic demonstrated the importance of robust and relatively inexpensive methods for the detection of RNA pathogens. Nucleic acid testing is the primary method for COVID-19 diagnosis. The testing involves the reverse transcription (RT) of severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) RNA into complementary DNA (cDNA) followed by amplification of target cDNA regions. A number of nucleic acid amplification techniques were developed for the rapid and sensitive detection of the virus [1], including reverse transcription polymerase chain reaction (RT-PCR) which is the “gold standard” for molecular diagnostic assays and the method predominantly employed for diagnosing COVID-19 using respiratory tract samples [2,3]. In general, RT-PCR and reverse transcription quantitative polymerase chain reaction (RT-qPCR) are the most important methods for the detection and quantification of target RNA sequences that are widely used for monitoring expression levels of mRNA and RNA biomarkers and the assay of RNA pathogens such as RNA viruses. One key aspect of efficient nucleic acid amplification are enzymes used in the reactions [4]. To aid in the development of diagnostic kits for rapid and sensitive detection of RNA pathogens, we evaluated novel engineered thermostable DNA polymerases with reverse transcriptase activity in RT-PCR assays.

Traditionally, RT-PCR is performed with two enzymes: a reverse transcriptase (e.g., MMLV or AMV revertase) for cDNA synthesis and a thermostable DNA-dependent DNA polymerase (e.g., Taq or Tth polymerase) for the PCR amplification of cDNA. However, using two separate enzymes complicates a reaction system and increases the cost of RT-PCR. Additionally, reverse transcriptases inhibit Taq polymerase activity and decrease the efficiency of PCR amplification [5,6,7,8]. These problems could be solved by using a single enzyme in a coupled RT and PCR amplification. Unfortunately, natural reverse transcriptases are not thermostable and cannot be used for PCR. On the contrary, Taq polymerase has very low RT activity and cannot be used for cDNA synthesis under normal conditions. The first attempts to tackle these problems were made in the 1990s when Mn^2+^ was used instead of Mg^2+^ to stimulate RT activity of Taq and Tth DNA polymerases [9,10,11,12]. Unfortunately, this improvement required routine optimization of the reaction buffer and did not provide high efficiency of the RT step of the reaction. Another approach to creating a single enzyme that could be used in RT and PCR was based on the protein engineering of the thermostable Family A (PolA) DNA polymerases, mainly Taq DNA polymerase. Several research groups have developed a selection of enzymes using different modifications of thermostable DNA polymerases, which are suitable for performing RT-PCR in a single enzyme mode.

The first articles describing modifications of Taq polymerase for enhancing reverse transcriptase activity were published in 2006 [13,14,15]. The most suitable enzymes for RT-PCR applications were mutants of Taq polymerase, which were described in 2010 by Prof. Andreas Marx and his research group from the University of Konstanz, Germany [16,17]. The optimized mutant of Taq polymerase contained four substitutions in the polymerase domain: S515R, L459M, I638F, and M747K. This development was commercialized by myPOLS Biotec GmbH (Germany) as the RevTaq DNA polymerase and Volcano master mix. The created polymerase was successfully applied for the detection of SARS-CoV2 RNA [18,19].

The scientists affiliated with QIAGEN (Beverly, MA, USA) created Magma DNA polymerase with an improved reverse transcriptase activity [20]. Magma polymerase is a fusion enzyme between the viral 3173 PyroPhage DNA polymerase and the 5′→3′ nuclease domain of Taq DNA polymerase. PyroPhage DNA polymerase is a thermostable viral Family A polymerase with inherent reverse transcriptase activity. The conjugation of the PyroPhage polymerase with the 5′→3′ nuclease domain of Taq DNA polymerase confers compatibility with TaqMan probes. Experiments demonstrated that Magma could be used to carry out RT-PCR [20]; however, this enzyme has not been commercialized.

Another modification of Taq polymerase was described in 2021 by Prof. Wayne Barnes and his colleagues from DNA Polymerase Technology, Inc. (St. Louis, MO, USA). They found that a single substitution D732N in the polymerase domain of Taq polymerase conferred strand displacement and reverse transcriptase activities on the enzyme [21]. The presence of strand displacement activity allows the enzyme to overcome the secondary structure of the RNA template; all natural reverse transcriptases possess a strong strand displacement activity [22]. The D732N mutant of Taq polymerase was commercialized by DNA Polymerase Technology, Inc. as the OmniTaq2 DNA polymerase.

A new variant of Tth DNA polymerase with reverse transcriptase activity was created by introducing two amino acid substitutions I709K and I640F into the enzyme [23].

The ReverHotTaq DNA polymerase, offered by Bioron GmbH (Römerberg, Germany), is another thermostable enzyme with strand displacement and reverse transcriptase activities. According to the description of the ReverHotTaq, it was obtained by incorporation of the fragments from the Bst DNA polymerase amino acid sequence into the active site of Taq DNA polymerase. The Bst polymerase has both strand displacement and a reverse transcriptase activities but is not thermostable enough for performing PCR [4]. Thus, the engineered ReverHotTaq combines the advantages of both parent enzymes: strand displacement and reverse transcriptase activities from the Bst polymerase as well as the 5′→3′ DNA polymerase and 5′→3′ exonuclease activities with high thermostability from the Taq polymerase.

The use of a single enzyme for coupled RT-PCR could greatly simplify the reaction system and decrease the cost of RT-PCR tests. Unfortunately, only a few examples of practical application of the modified thermostable polymerases for RT-PCR tests are currently known in the field [18,19] and a direct comparison of the enzymes inter se has not yet been performed.

In the present work, we compare three different commercially available thermostable DNA polymerases with reverse transcriptase activity (RevTaq, OmniTaq2, and ReverHotTaq) by performing end-point and real-time RT-PCR assays of SARS-CoV2 RNA and mRNA molecules. Conventional M-MLV/Taq polymerase mixtures for OneTube RT-PCR were used as standards.

2. Materials and Methods

2.1. Enzymes and Reagents

The following materials were obtained from these sources: 100X OmniTaq2 DNA polymerase and 10X Taq Mutant Reaction Buffer from DNA Polymerase Technology, Inc. (St. Louis, MO, USA); 50X RevTaq DNA polymerase and 5X Volcano Reaction Buffer from myPOLS Biotec GmbH (Konstanz, Germany); 50X ReverHotTaq polymerase and 5X Reaction Buffer from Bioron GmbH (Römerberg, Germany); OneTaq One-Step RT-PCR Kit from New England BioLabs, Inc. (Ipswich, MA, USA); 5X OneTube RT-PCR-Mix from Evrogen (Moscow, Russia); and dNTPs from Bioline Limited (London, UK). Oligonucleotide primers and TaqMan probes were synthesized by Syntol JSC (Moscow, Russia). SARS-CoV-2 viral RNA (2500 copies/µL) isolated from nasopharyngeal swabs was provided by Syntol JSC and verified by RT-qPCR with positive synthetic SARS-CoV-2 RNA Control 2 (SKU: 102024) from Twist Bioscience (San Francisco, CA, USA) and SARS-CoV-2 RT-qPCR Detection Kit from Syntol JSC (Moscow, Russia). The real-time amplification reactions were performed using a CFX96 Touch Real-Time Detection System (Bio-Rad Laboratories, Inc., Hercules, CA, USA). EvaGreen intercalating dye was supplied by Syntol JSC (Moscow, Russia).

Human total RNA was isolated from human embryonic kidney 293 (HEK 293) cells by using TRIzol reagent (ThermoFisher Scientific, Inc., Carlsbad, CA, USA) and then subjected to treatment with RNase-Free DNase I (RNase-Free DNase Set, Qiagen GmbH, Hilden, Germany), followed by column purification (RNeasy Mini Kit, Qiagen GmbH, Hilden, Germany). Total RNA concentration was measured with a Qubit RNA High Sensitivity kit and Qubit-4 fluorimeter (ThermoFisher Scientific, Inc., Carlsbad, CA, USA).

2.2. Coupled End-Point RT-PCR

End-point RT-PCR assays were carried out with 10 ng, 1 ng, 100 pg, or 10 pg of human total RNA or without RNA as a negative control. The reaction mixtures (25 µL) contained 0.25 mM dNTP (each), two primers (0.3 µM, each), 1X concentration of RevTaq (0.5 µL) or OmniTaq2 (0.25 µL) or ReverHotTaq (0.5 µL) or One Taq One-Step Enzyme Mix (1 µL), and 1X concentration of the appropriate reaction buffer. The following primers were used for the amplification of beta-2-microglobulin cDNA fragments: B2M-f1 (5′-CTGCCGTGTGAACCATGTGA) and B2M-r1 (5′-CAATCCAAATGCGGCATCTTC) for the 105 bp amplicon; B2M-f2 (5′-TGTAAGCAGCATCATGGAGGTT) and B2M-r2 (5′-TGCTCAGATACATCAAACATGG) for the 203 bp amplicon; B2M-f1 and B2M-r3 (5′-CTCTGCTCCCCACCTCTAAGTT) for the 317 bp amplicon.

The following conditions were used for end-point RT-PCR with RevTaq, OmniTaq 2, and ReverHotTaq DNA polymerases: (a) initial denaturation at 75 °C for 2 min; (b) reverse transcription at 60 °C for 10 min, then 68 °C for 30 min; (c) denaturation at 92 °C for 1 min; (d) PCR amplification by 38 thermocycles at 92 °C for 30 s, 60 °C for 30 s, and 68 °C for 30 s. For RT-PCR with the OneTaq One-Step RT-PCR enzyme mixture, the following conditions were applied: (a) RT at 48 °C for 30 min; (b) denaturation at 92 °C for 1 min; (c) PCR amplification by 38 thermocycles at 92 °C for 30 s, 60 °C for 30 s, and 68 °C for 30 s.

2.3. Coupled Real-Time RT-qPCR with an Intercalating Dye

Real-time RT-qPCR assays with EvaGreen DNA-binding dye were carried out with 1 ng, 100 pg, or 10 pg of human total RNA or without RNA as a negative control. Polymerases RevTaq, OmniTaq 2, ReverHotTaq, and OneTube RT-PCRMix were used for the assays. The reaction mixtures were identical to the mixtures of end-point RT-PCR described above, except the primers for target amplification. Additionally, the reaction mixtures contained 0.4X EvaGreen DNA-binding dye. Coupled reverse transcriptions and real-time amplifications of a 109 bp fragment of human GAPDH cDNA were performed with primers GAPDH_d (5′-GTGGTCTCCTCTGACTTCAACAG) and GAPDH_r (5′-CGTTGTCATACCAGGAAATGAGCTTG).

For RT-qPCR with RevTaq, OmniTaq 2, and ReverHotTaq polymerases, we used the following conditions: (a) initial denaturation at 75 °C for 2 min; (b) reverse transcription at 62 °C for 10 min, then 68 °C for 15 min; (c) denaturation at 92 °C for 2 min; (d) PCR amplification by 40 thermocycles: 92 °C for 15 s, 60 °C for 15 s, and 68 °C for 15 s. For RT-qPCR with OneTube RT-PCRmix, we applied (a) reverse transcription at 50 °C for 20 min; (b) denaturation at 92 °C for 2 min; (c) PCR amplification by 40 thermocycles: 92 °C for 15 s, 60 °C for 15 s, and 68 °C for 15 s.

2.4. Coupled Real-Time RT-qPCR with a TaqMan Probe

TaqMan probe-based RT-qPCRs targeting SARS-CoV-2 viral RNA were performed using OneTube RT-PCRMix and polymerases: RevTaq, OmniTaq 2, and ReverHotTaq. The reactions were carried out over a 5-log range of input template RNA concentrations. The RNA isolated from nasopharyngeal swabs containing SARS-CoV-2 viral genomic RNA (gRNA) was diluted 10, 50, and 250 times and used as a template. The reaction mixtures (25 µL) contained 10 µL of the template RNA (2500, 500 or 100 copies per reaction), 0.25 mM dNTP (each), two primers (0.4 µM, each), TaqMan probe (0.2 µM), 1X concentration of RevTaq (0.5 µL) or OmniTaq2 (0.25 µL) or ReverHotTaq (0.5 µL) or OneTube RT-PCRMix (5 µL), and 1X concentration of the appropriate reaction buffer. The following primers and probe were used for the RT-qPCR reactions: CoV-d (5′-ACCTTAAATTCCCTCGAGGACAA), CoV-r (5′-TAGGTAGTAGAAATACCATCTTGGACTGAGA), and CoV-probe (5′-(ROX)-TCCAATTAACACCAATAGCAGTCCAGA-(BHQ2)).

The assays with RevTaq, OmniTaq 2, and ReverHotTaq polymerases were performed as follows: (a) initial denaturation at 75 °C for 2 min; (b) reverse transcription at 62 °C for 10 min, then 68 °C for 15 min; (c) denaturation at 92 °C for 2 min; (d) PCR amplification by 50 thermocycles: 92 °C for 15 s, 58 °C for 15 s, and 70 °C for 15 s. The conditions for RT-qPCR with OneTube RT-PCRmix are as follows: (a) reverse transcription at 50 °C for 20 min; (b) denaturation at 92 °C for 2 min; (c) PCR amplification by 50 thermocycles: 92 °C for 15 s, 58 °C for 15 s, and 70 °C for 15 s.

2.5. Data Analysis

All calculations were performed using R software [24]. Core R language and several extension packages were used, including RDML for raw amplification data manipulation [25], chipPCR for Cq calculation [26], and ggplot2 for the generation of plots [27].

3. Results

3.1. End-Point RT-PCR

The performance of three thermostable DNA polymerases with reverse transcriptase activity (RevTaq, OmniTaq2, and ReverHotTaq) was tested in end-point RT-PCR assays. OneTaq One-Step Enzyme Mix (NEB), a specially designed enzyme mixture for end-point RT-PCR, was used as a standard. Target sequences of human beta-2-microglobulin (B2M) mRNA of different sizes (105 bp, 203 bp and 317 bp) were amplified as described in Section 2. For the experiments, we used total human RNA and primers for B2M mRNA. As the human genome does not contain any pseudogenes of B2M, it is widely used as the endogenous reference gene for the relative quantification of genes of interest [28].

Figure 1 shows that all the enzymes demonstrated similar sensitivity and product yields when amplifying the shortest 105 bp fragment. The efficiency of the RevTaq and ReverHotTaq polymerases was equal to that of the OneTaq One-Step RT-PCR Kit when amplifying the 203 bp fragment. However, the results for the OmniTaq2 polymerase showed that once the length of the cDNA fragments increased from a small 105 bp fragment to a larger 203 bp or 317 bp fragment, the yield of the specific products decreased. When amplifying the largest 317 bp fragment, a ten-fold decrease in sensitivity was demonstrated by RevTaq, OmniTaq2, and ReverHotTaq polymerases compared to the OneTaq One-Step RT-PCR Kit (Figure S1 and Table S1). Figure 1 also shows that in all reactions, RevTaq polymerase generates more visible amounts of non-specific products, compared to other enzymes.

3.2. Real-Time RT-qPCR with an Intercalating Dye

The performance of RevTaq, OmniTaq2, and ReverHotTaq DNA polymerases was compared to that of the OneTube RT-PCRMix in real-time RT-qPCR with EvaGreen dye. The OneTube RT-PCRMix containing a reverse transcriptase and Taq polymerase is a specially designed enzyme mixture for coupled real-time RT-qPCR. The target 109 bp sequence of human GAPDH mRNA was amplified from the series of dilutions of human total RNA as described in the Section 2. GAPDH is the standard endogenous reference gene for expression studies [28]. Figure 2 and Table 1 show the results of the RT-qPCRs; ReverHotTaq DNA polymerase and OneTube RT-PCRMix had similar efficiency with different template concentrations. RevTaq polymerase showed similar results to ReverHotTaq when 1 ng and 100 pg of the total RNA were used but generated non-specific products at low template concentration (Figure 2B, melting curves), which prevented the detection of GAPDH mRNA in 10 pg of human total RNA (Figure 2B). OmniTaq2 DNA polymerase allowed the RT-qPCR assay to be carried out with all the RNA template concentrations (Figure 2C) but demonstrated significantly lower efficiency of amplification than the other enzymes, which was seen as a 3–6 cycle lag in the RT-qPCR (Figure 2, Table 1). In the no template control (NTC) reactions, all enzymes generated signals from non-specific products (Figure 2).

3.3. Real-Time RT-qPCR with a TaqMan Probe

RevTaq, OmniTaq2, and ReverHotTaq DNA polymerases were compared with OneTube RT-PCRMix by performing coupled RT-qPCR assays with a hydrolysis TaqMan probe. The target sequence of SARS-CoV-2 RNA was detected in the sample of nasopharyngeal swab RNA containing SARS-CoV-2 viral gRNA. The reactions were carried out with the series of dilutions of the RNA template (2500, 500, or 100 copies of the template per reaction), as described in the Section 2. The results of the assays demonstrated that RevTaq, ReverHotTaq, and OneTube RT-PCRMix had similar efficiency in RT-qPCR with the TaqMan probe and were able to detect at least 100 copies of SARS-CoV-2 gRNA per reaction (Table 1 and Figure 3). OmniTaq2 DNA polymerase did not generate a fluorescent signal with the TaqMan probe and failed in this test (Figure 3D).

4. Discussion

RT-PCR and RT-qPCR are widely used methods for the detection of target RNA sequences, including viral RNAs and endogenous mRNAs. In contrast to traditional enzymatic mixtures for RT-PCR that contain a reverse transcriptase and a thermostable DNA polymerase, the modified thermostable RevTaq, OmniTaq2, and ReverHotTaq DNA polymerases with reverse transcriptase activity could be used as single enzymes for RT-PCR. These artificial polymerases were created by different modifications of Taq DNA polymerase, and therefore, contain different amino acid substitutions. We compared the efficiency of the modified DNA polymerases to the conventional enzyme mixtures for RT-PCR in end-point RT-PCR and real-time RT-qPCR using B2M and GAPDH mRNAs as the target RNA sequences. These two genes are commonly used for expression studies [28] (Balaji, S. et al., 2020). SARS-CoV2 virus RNA, which was an actual specimen of the viral pathogen, was used as the target RNA template. We used primers and probes that were designed for in-house routine RT-PCR experiments and optimized for applications with conventional RT-PCR enzyme mixtures.

At the end-point of the RT-PCR experiments, the modified DNA polymerases were compared with the specially designed enzyme mixture for end-point RT-PCR—OneTaq One-Step Enzyme Mix (NEB). We amplified different fragments of the B2M mRNA sequence and found that the increasing length of the amplified sequence from 105 to 317 bases decreased the sensitivity of the assays with the modified DNA polymerases in contrast to the standard One-Step Enzyme Mix. This might indicate that the traditional reverse transcriptase from the RT-PCR Kit has better performance with respect to the long RNA fragments than the currently available, modified DNA polymerases. Furthermore, the amplifications with RevTaq polymerase generated visible amounts of non-specific products, in contrast to the other enzymes. The appearance of non-specific products could be the result of the relatively low annealing temperature of the used primers (60 °C). Usually, a high melting temperature is recommended for the primers (>65 °C) used with RevTaq polymerase.

In the real-time RT-qPCR experiments, we compared the modified DNA polymerases with the OneTube RT-PCR Enzyme Mixture specifically designed for coupled RT-qPCR with intercalating dyes or TaqMan probes. When we used EvaGreen dye in the real-time assays, the OneTube RT-PCR Enzyme Mixture, the RevTaq, and the ReverHotTaq polymerases showed better performance than the OmniTaq2. However, the RevTaq polymerase tended to generate non-specific products at low template concentration that prevented target sequence detection. As mentioned above, this could be the result of the relatively low annealing temperature of the used primers that were originally optimized for the conventional OneTube RT-PCRMix containing reverse transcriptase.

Real-time RT-qPCR with a hydrolysis TaqMan probe is the main technique for the molecular detection of RNA pathogens, including the SARS-CoV-2 virus. We demonstrated that in SARS-CoV-2 RT-qPCR assays with the TaqMan probe, the RevTaq and ReverHotTaq polymerases could be used for the detection of the viral RNA on par with the conventional OneTube RT-PCR Enzyme Mixture. Moreover, RevTaq has been successfully used for SARS-CoV-2 RNA detection in raw patient samples [18] and wastewater [19] and demonstrated the LOD (limit of detection) five–ten copies of the viral genome equivalent [18]. The OmniTaq2 DNA polymerase did not generate any fluorescent signal with the TaqMan probe. It could be possible that the OmniTaq2 had a reduced 5′→3′ exonuclease activity in our experiments and could not efficiently hydrolyze the TaqMan probe.

When comparing the modified DNA polymerases and traditional enzyme mixtures suited for RT-PCR, the economic advantages of using the modified DNA polymerases should be mentioned. Thus, the estimated cost of the artificial thermostable DNA polymerases with reverse transcriptase activity per reaction does not exceed 1 EUR (RevTaq approximately 1 EUR, OmniTaq2 approximately 0.7 USD, and ReverHotTaq 0.76 EUR), which is significantly cheaper than traditional enzyme mixtures for RT-PCR.

5. Conclusions

The artificial thermostable DNA polymerases with reverse transcriptase activity can be used as simple and relatively inexpensive tools for the detection of target RNAs by coupled RT-PCR and RT-qPCR with the same time and labor costs as traditionally used reagents. Comparison to the traditional enzyme mixtures for RT-PCR containing a reverse transcriptase showed the tested polymerases have a lower efficiency of long-fragment RT-PCR amplification. Thus, these artificial polymerases are better suited for the efficient amplification of short (up to 200 bases) RNA sequences, which would be sufficient for a wide spectrum of RT-PCR assays, including the detection of RNA-based pathogens, e.g., SARS-CoV2. The RevTaq, OmniTaq2, and ReverHotTaq polymerases could be used for end-point RT-PCR and real-time RT-qPCR with an intercalating dye. Real-time RT-qPCR with a hydrolysis TaqMan probe could be carried out with the RevTaq and ReverHotTaq polymerases. Based on our data we can conclude that the commercially available artificial thermostable DNA polymerases with reverse transcriptase activity provide a robust and cheap alternative to the traditional approaches in the RT-PCR field.