Benchmarking viral RNA amplification performance with the GoTaq® Probe 1-Step RT-qPCR System
Promega Corporation
Publication Date: 11/2017
Abstract
In this article, we demonstrate that GoTaq® Probe 1-Step RT-qPCR System (Cat.# A6120) performs, in singleplex and multiplex, as well as or better than leading competitors with two viral targets.
GoTaq® Probe 1-Step RT-qPCR System (Cat.# A6120) is a flexible tool for one step combined reverse transcription and qPCR using hydrolysis probes. This system works for a multitude of applications from simple gene expression measures to complex multiplexing. Here we demonstrate the versatility and performance of the system with two viral models. We will compare the performance of this system with three competitors: SuperScript® III Platinum One-Step qRT-PCR, Verso™ 1-Step RT-qPCR, and Prime Script™ One-Step RT-PCR Kits. GoTaq® 1-Step RT-qPCR performed as well as or better than leading competitors with the viral targets tested.
Materials and Methods
Viral amplification was performed with each master mix using 4-fold dilutions from 50,000 copies down to 12 copies of HIV and Zika RNA (ATCC Cat.# VR-3245SD and VR-3252SD). Singleplex and multiplex reactions were performed in quadruplicate with standard cycling conditions recommended by each product's technical manual. Reaction volumes were 25µl for all master mixes with a final primer concentration of 400nM and probe concentration of 200nM. Primers and hydrolysis probes were synthesized by Integrated DNA Technologies in their PrimeTime® qPCR Assay format and sequences are listed below. Reactions were performed on an Applied Biosystems® 7500 Fast Real-Time PCR System.
Zika Primer/Probe Set Sequence¹:
Forward: CCG CGT CCC AAC ACA AG
Reverse: CCA CTA ACG TTC TTT TGC AGA CAT
Probe: 5' AGC CTA CCT TGA CAA GCA GTC AGA CAC TCA A 3' 6-FAM/ZEN/IBFQ
HIV Primer/Probe Set Sequence:
Forward: TGC AGA ATG GGA TAG ATT GC
Reverse: CCC TTG GTT CTC TCA TCT GG
Probe: 5' CCT GGT GCA ATA GGC CCT CCA 3' 6-VIC/ZEN/IBFQ
Results
We tested GoTaq® Probe 1-Step qPCR System for amplification efficiency, linearity and sensitivity against three leading competitors. HIV and Zika RNAs were amplified using the three mixes tested in singleplex (Figure 1) and as multiplexed reactions (Figure 2). Ideally, when multiplexing there will be less than one Cq difference between multiplex and singleplex reactions to ensure no interference is occurring between the assays, which can cause problems with target quantitation. GoTaq®, PrimeScript™, and Verso™ master mixes showed less than one Cq difference with RNA amplified in multiplex or singleplex. SuperScript® III Platinum exhibited greater than one Cq difference between reactions. Standard curve linearity and efficiency for single and multiplex reactions are listed for each master mix in Table 1. GoTaq® demonstrated excellent linearity (≥0.995), sensitivity (down to 12 viral copies), and efficiency (≥97.0%). All competitors had similar standard curve R² values and efficiencies, especially with multiplexes (Table 1). These results suggest that GoTaq® Probe 1-Step RT-qPCR System can perform equivalently or better than the other one step RT-qPCR master mixes tested.
Figure 1. Amplification of viral RNA with different master mixes. Serially diluted control RNA from HIV Virus (right) or Zika Virus (left) was amplified using GoTaq® Probe 1-Step RT-qPCR System or three other competitor products.
Figure 2. Sensitivity with singleplex or multiplex reactions. RNA from Zika Virus was amplified in single or multiplex with HIV using either GoTaq® Probe 1-Step RT-qPCR System (A) or competitor master mixes (B-D).
Table 1. Efficiency and linearity of viral RNA amplification in single and multiplex. Viral RNA standard curves were amplified in single or multiplex using GoTaq® Probe 1-Step RT-qPCR System or competitor kits. Linearity (R²) and efficiency (%) were determined from standard curves using the Applied Biosystems® 7500 software.
Conclusions
GoTaq® Probe 1-Step RT-qPCR System is a flexible system for amplification for a diverse range of targets, including viruses. It also can be used for the detection of multiple targets in a single reaction with no loss in efficiency or linearity. GoTaq® Probe 1-Step RT-qPCR System is a robust, convenient master mix for one-step reverse transcription qPCR.
References
¹Lanciotti RS, Kosoy OL, Laven JJ, et al. Genetic and Serologic Properties of Zika Virus Associated with an Epidemic, Yap State, Micronesia, 2007. Emerging Infectious Diseases. 2008;14(8):1232-1239.
a)U.S. Pat. No. 6,242,235, Australian Pat. No. 761757, Canadian Pat. No. 2,335,153, Chinese Pat. No. ZL99808861.7, Hong Kong Pat. No. HK 1040262, Japanese Pat. No. 3673175, European Pat. No. 1088060 and other patents pending.
(b)NOTICE TO PURCHASER: DISCLAIMER OF LICENSE
GoTaq® Probe 1-Step RT-qPCR System (Cat.# A6120) is for Research Use Only. Not for use in Diagnostic Procedures.
No license is conveyed with the purchase of this product under any of US Pat. Nos. 5,210,015, 5,487,972, 5,804,375, 5,994,056, 6,171,785, 6,214,979, 5,538,848, 5,723,591, 5,876,930, 6,030,787, and 6,258,569, and corresponding patents outside the United States, or any other patents or patent applications, relating to the 5’ Nuclease and dsDNA-Binding Dye Processes. For further information contact the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA.
© 2017 Promega Corporation. All Rights Reserved.
GoTaq is a registered trademark of Promega Corporation.
SuperScript is a registered trademark of Invitrogen Corporation.
Verso is a trademark of Thermo Fisher Scientific, Inc. PrimeScript is a trademark of Takara Bio Inc.
