Rutschke et al. Journal of Analytical Science and Technology (2015) 6:20
DOI 10.1186/s40543-015-0063-4
SHORT REPORT Open Access
Hot start reverse transcriptase: an approach for improved real-time RT-PCR performance
http://crossmark.crossref.org/dialog/?doi=10.1186/s40543-015-0063-4&domain=pdf
Web End = Nils Rutschke1,3*, Jan Zimmermann1, Ronny Mller1,3, Gerd Klck2, Mathias Winterhalter3 and Annika Leune1
Abstract
Background: Reverse transcriptase is an indispensable enzyme for real-time reverse transcriptase (RT)-PCR, a standard method in molecular diagnostics for detection and quantification of defined RNA molecules. The prevention of non-specific products due to elongation of misprimed oligonucleotides by the enzyme at temperatures beneath the specific annealing temperature is one of the biggest challenges in real-time RT-PCR.
In the present study, an aptamer directed against the reverse transcriptase was analyzed for its potential to attain a temperature-dependent reverse transcriptase (hot start RT).
Findings: The hot start effect was investigated in a one-step real-time RT-PCR assay for the detection of Middle East respiratory syndrome coronavirus (MERS-CoV). Results with aptamer revealed a reduced RT activity at low temperatures while achieving full activity at the specific annealing temperature of 55 C. Sensitivity (limit of detection (LoD) 95 %) of the MERS-CoV assay was increased by about two times in the presence of aptamer.
Conclusions: The study demonstrates the potential of aptamer-dependent hot start RT for the improvement of diagnostic real-time RT-PCR assays.
Keywords: Real-time RT-PCR; Reverse transcriptase; Hot start; MERS-CoV
Findings
IntroductionReal-time RT-PCR is the method of choice in molecular diagnostics for detection and quantification of defined RNA molecules (Mackay 2004). This technique utilizes reverse transcriptase (RT) to convert RNA into complementary DNA (cDNA), a thermostable DNA-dependent DNA polymerase for the amplification of specific target sequences and target specific probes (oligonucleotides) labelled with fluorophores for the detection of amplified DNA (Gibson et al. 1996).
Real-time RT-PCR is regarded as a method with high sensitivity and specificity (Martel et al. 2002). However, this is challenged by non-specific products generated by elongation of misprimed primer that competes with the synthesis of specific amplification products in each cycle (Chou et al. 1992; Li et al. 1990). The probability of non-specific product formation increases with the complexity
of the real-time RT-PCR system and the background nucleic acid in the specimen (Brownie et al. 1997, Handschur et al. 2009). Ultimately, non-specific products can severely decrease sensitivity as well as specificity of real-time RT-PCR assays (Sharkey et al. 1994; Birch et al. 1996; Jayasena 1999).
Assays for the detection of RNA viruses are often highly complex (high quantity of different oligonucleo-tides) due to low sequence conservation of the RNA genome (Gardner et al. 2004; Brownie et al. 1997). In general, mispriming occurs at temperatures below the specific annealing temperature of the oligonucleotides (Jayasena 1999). Thus, the formation of non-specific products can be reduced by using hot start variants of the enzymes, which are inactive at low temperatures and activated at higher temperatures, appropriate for specific primer annealing to the target nucleic acid (Birch et al. 1996).
Several biological or chemical hot start concepts exist for Taq polymerase, a thermostable DNA-dependent DNA polymerases. The Taq polymerase can be inactivated by binding of specific antibodies or aptamers, by incubation with chemicals or by altered molecular kinetics
* Correspondence: mailto:[email protected]
Web End [email protected]
1altona Diagnostics GmbH, Moerkenstr. 12, 22767 Hamburg, Germany
3School of Engineering and Science, Jacobs University, Campus Ring 1, 28759 Bremen, GermanyFull list of author information is available at the end of the article
2015 Rutschke et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0
Web End =http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited.
Rutschke et al. Journal of Analytical Science and Technology (2015) 6:20 Page 2 of 5
(Sharkey et al. 1994; Birch et al. 1996, Hermann and Patel 2000; Gening et al. 2006; Kermekchiev et al. 2003). The activation is obtained by heating up to 95 C during the initial denaturation step of the PCR. In contrast to Taq polymerase, which is heat-stable up to temperatures of 95 C, the RT is only stable at temperatures ranging from 42 to 70 C (Pfaffl 2010; Gallup 2011). Therefore, other hot start concepts for the reversible inactivation of reverse transcriptase need to be developed.
A high-affinity RNA ligand (aptamer), which targets moloney murine leukemia virus (M-MLV) RT was described by Chen and Gold (1994). The aptamer is assumed to inactivate the RT by blocking the nucleic acid binding site of the RT (Chen and Gold 1994; Chen et al. 1996).
In the present study, the aptamer was analyzed in a one-step real-time RT-PCR assay for the detection of Middle East respiratory syndrome coronavirus (MERSCoV) to investigate the potential of a hot start RT for improved real-time RT-PCR performance. MERS-CoV was first identified in 2012 (Zaki et al. 2012). Since the discovery, the World Health Organization noted 971 cases of MERS-CoV, and of these, 365 led to a lethal outcome (WHO 2015). Therefore, a more sensitive detection system, especially in the presence of low viral loads in patients, would be favorable for an early diagnosis and treatment.
Material and methodsReal-time RT-PCR setupThe one-step real-time RT-PCR was performed in a 25-L reaction mix containing 10 L of RNA template, 1x PCR reaction buffer (altona Diagnostics GmbH), 2.4 mM MgCl2 (Sigma-Aldrich), 240 g/L BSA (Roche), 1 U of Platinum Taq DNA Polymerase high fidelity (Invitrogen), 156 U of SuperScript III Reverse Transcript-ase (Invitrogen).
The MERS-CoV specific primer and probe, targeting the genomic region upstream of the Envelope gene (upE), were synthesized as published (Corman et al. 2012a; Corman et al. 2012b). The RT-PCR reaction included 0.8 M of primer UpE-Fwd (GCAACGCGCGATTCAGTT), 0.8 M of primer UpE-Rev (GCCTCTACACGGGACCCATA), and0.1 M of probe (FAM-CTCTTCACATAATCGCCCCGA GCTCG-TAMRA).
Thermal cycling conditions were 55 C for 20 min (RT-step), 2 min at 95 C (Taq polymerase activation and RT inactivation), followed by 45 cycles of 15 s at 95 C (denaturation), 45 s at 58 C (annealing and acquisition) and 15 s at 72 C (extension). In case of thermal gradient real-time RT-PCR, the RT-step was performed in parallel at 31 C for one part and 55 C for the other part of the 96-well plate.
All real-time RT-PCRs were performed on a CFX96 Touch Real-Time PCR Detection System (BioRad).
Fig. 1 Relation between aptamer concentration and cycle threshold (Ct) at 31 and 55 C. Aptamer concentrations of 0, 12.5, 25, and 50 M per
reaction (a) and 0, 50, 100, and 200 M per reaction (b) were tested in a one-step real-time RT-PCR assay for the detection of Middle East respiratory
syndrome coronavirus (MERS-CoV) using 1000 RNA copies per reaction. RT-step was carried out for 20 min in parallel at 31 C (circles) and 55 C (dots),
respectively. Results of 200 M aptamer concentration at 31 C did not lead to a positive signal and therefore are not shown
Rutschke et al. Journal of Analytical Science and Technology (2015) 6:20 Page 3 of 5
Table 1 Hit rate of 25 M aptamer and without aptamer in real-time RT-PCR MERS-CoV assay. Half-logarithmic serial dilutions of MERS-CoV RNA, ranging from 101 to 102 copies per reaction were analyzed. The ratio of positive signals to all signals, followed by the data in percentage is given below
Copies (MERS-CoV IVT)/reaction102 101.5 101 100.5 100 100.5 101
Aptamer (25 M/reaction) 24/24 24/24 24/24 19/24 11/24 2/24 1/24
100 % 100 % 100 % 79.17 % 45.83 % 8.33 % 4.17 % Control (without aptamer) 24/24 24/24 22/24 13/24 5/24 2/24 0/24
100 % 100 % 91.67 % 54.17 % 20.83 % 8.33 % 0 %
AptamerThe aptamer (5-CUUACCACGCGCUCUUAACUGCUA GCGCCAUGGCCAAAACU-3) published by Chen and Gold (1994) was synthesized at altona Diagnostics GmbH. A 3 phosphorylation was added to the aptamer to eliminate any possible elongation of the aptamer.
The reverse transcriptase was incubated with increasing concentrations (0, 12.5, 25, 50, 100, or 200 M) of aptamer for 15 min at room temperature (20 C) before adding to the reaction mix.
To demonstrate an aptamer-dependent hot start RT effect, the RT-step was carried out in parallel at 55 C, the specific annealing temperature of the primer and probe, and at 31 C, at which the RT shows a significant activity but which is below the specific annealing temperature
of the primer and probe. Each aptamer concentration was analyzed in three replicates.
Real-time RT-PCR templateAn in vitro transcribed RNA (IVT) based on a sequence of MERS-CoV strain EMC/2012 was used as real-time RT-PCR template. The concentration of the IVT was determined by spectrophotometry.
Valuation of analytic sensitivityThe analytic sensitivity (limit of detection (LoD)) is defined as the concentration (copies/reaction) of MERSCoV specific RNA (IVT) molecules that can be detected with a positive rate of 95 %. The analytic sensitivity was determined by analyzing a half-logarithmic serial dilution
Without Aptamer 25 M Aptamer
A B
Fig. 2 Probit regression analyses of a real-time RT-PCR with 25 M aptamer and without aptamer. Probit regression analysis was carried out with
target RNA loads from 101 to 102 copies per reaction. Each dilution was analyzed in four independent runs with six replicates. The target RNA
concentration is plotted on the X-axis, and the Y-axis displays the hit rate. Circles are experimental data points; the inner lines represent the
corresponding probit curve, outer lines the 95 % confidence intervals. a Without aptamer; b 25 M aptamer
Rutschke et al. Journal of Analytical Science and Technology (2015) 6:20 Page 4 of 5
of the IVT ranging from 100 to 0.1 copies/reaction. Each concentration was analyzed four times in six replicates (n = 24). Hit rates were subjected to probit regression and correlation analysis in StatsDirect software (Version 2,7,9; StatsDirect statistical software).
The RT was either incubated with 25 M aptamer or without aptamer for 15 min at room temperature (20 C), before adding to the reaction mix.
ResultsAptamer leads to hot start effectThe reverse transcriptase was incubated with different concentrations of the aptamer (0, 12.5, 25, 50, 100, and 200 M per reaction) before adding to the reaction mix. The RT-step was performed in parallel at 55 and 31 C.
Incubation of RT with aptamer and RT-step at 31 C resulted in a mean delayed cycle threshold of up to Ct 7.31 (50 M aptamer, Fig. 1a) compared to the reference without aptamer, indicating reduced RT activity (Fig. 1a, b).
The results can be summarized as, the higher the aptamer concentration the later the amplification signal. Two hundred micromolar of aptamer revealed no amplification signal at all.
The RT-step at 55 C, with 12.5 and 25 M aptamer showed slightly earlier cycle thresholds compared to the reference without aptamer (Ct 0.60 and 0.61), while concentrations of 50 M and higher resulted in delayed amplification signals, compared to the reference without aptamer (Fig. 1a, b).
Since the aptamer concentration of 25 M allowed full RT activity at 55 C but significantly reduced RT activity at 31 C, this concentration was chosen to survey the influence of the aptamer on the analytic sensitivity of the MERS-CoV assay.
Evaluation of hot start RT for improved detection of a low copy number targetIn order to investigate, if the hot start RT lead to an increase in analytical sensitivity of the MERS-CoV assay, a half-logarithmic serial dilution of the MERS-CoV IVT ranging from 100 to 0.1 copies per reaction was analyzed with a reaction mix containing hot start RT (25 M aptamer) and standard RT (without aptamer).
Real-time RT-PCR was carried out to determine concentration-dependent hit rates (Table 1), which were analyzed in a probit regression (Fig. 2).
The MERS-CoV assay with the hot start RT has a LoD of 6.9 copies/reaction (95 % confidence interval (CI):4.215.4 copies/reaction), whereas the assay without aptamer has a LoD of 15.5 copies/reaction (95 % confidence interval (CI): 9.334.7 copies/reaction) (Fig. 2a, b).
ConclusionHot start Taq polymerases have proven to be valuable tools to improve analytical sensitivity and specificity in real-time PCR assays, by reducing non-specific products.
Based on this experience, the idea arose to improve the performance of real-time RT-PCR assays by developing a hot start concept for the reverse transcriptase.
In this study, we demonstrated that a hot start RT can be generated by using an aptamer directed against M-MLV RT.
The use of this hot start RT in a MERS-CoV assay led to an increase of the analytical sensitivity of about twofold compared to the analytical sensitivity of the same assay with standard RT.
In summary, we could demonstrate that hot start RT has the potential to improve the sensitivity of real-time RT-PCR assays.
This finding will be of special interest for more complex assays or for assays which are used with specimen with a high load of non-target nucleic acids in routine molecular diagnostics.
Competing interestsAuthors NR, AL, RM, and JZ are employees of altona Diagnostics GmbH. The authors declare that they have no competing interests.
Authors contributionsNR has performed the experimental and analytical work and prepared the draft of the manuscript. JZ provided oligos and aptamers. RM involved in experiment design and data analysis. The guidelines and supervision of this work was provided by AL and GK. AL and MW read and modified the manuscript. All authors read and approved the final manuscript.
Author details
1altona Diagnostics GmbH, Moerkenstr. 12, 22767 Hamburg, Germany.
2Institute of Environmental Biology and Biotechnology, University of Applied Sciences Bremen, Am Neustadtswall 30, 28199 Bremen, Germany. 3School of Engineering and Science, Jacobs University, Campus Ring 1, 28759 Bremen, Germany.
Received: 8 June 2015 Accepted: 14 June 2015
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The Author(s) 2015
Abstract
Background
Reverse transcriptase is an indispensable enzyme for real-time reverse transcriptase (RT)-PCR, a standard method in molecular diagnostics for detection and quantification of defined RNA molecules. The prevention of non-specific products due to elongation of misprimed oligonucleotides by the enzyme at temperatures beneath the specific annealing temperature is one of the biggest challenges in real-time RT-PCR.
In the present study, an aptamer directed against the reverse transcriptase was analyzed for its potential to attain a temperature-dependent reverse transcriptase ("hot start" RT).
Findings
The hot start effect was investigated in a one-step real-time RT-PCR assay for the detection of Middle East respiratory syndrome coronavirus (MERS-CoV). Results with aptamer revealed a reduced RT activity at low temperatures while achieving full activity at the specific annealing temperature of 55 °C. Sensitivity (limit of detection (LoD) 95 %) of the MERS-CoV assay was increased by about two times in the presence of aptamer.
Conclusions
The study demonstrates the potential of aptamer-dependent hot start RT for the improvement of diagnostic real-time RT-PCR assays.
You have requested "on-the-fly" machine translation of selected content from our databases. This functionality is provided solely for your convenience and is in no way intended to replace human translation. Show full disclaimer
Neither ProQuest nor its licensors make any representations or warranties with respect to the translations. The translations are automatically generated "AS IS" and "AS AVAILABLE" and are not retained in our systems. PROQUEST AND ITS LICENSORS SPECIFICALLY DISCLAIM ANY AND ALL EXPRESS OR IMPLIED WARRANTIES, INCLUDING WITHOUT LIMITATION, ANY WARRANTIES FOR AVAILABILITY, ACCURACY, TIMELINESS, COMPLETENESS, NON-INFRINGMENT, MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE. Your use of the translations is subject to all use restrictions contained in your Electronic Products License Agreement and by using the translation functionality you agree to forgo any and all claims against ProQuest or its licensors for your use of the translation functionality and any output derived there from. Hide full disclaimer