An increasing number of specialized real-time qPCR master mixes claiming to be specially designed for detecting certain targets have entered the genomics market. When looking to design a new assay or switch products, it might appear that these products would give a higher likelihood of success, but is this really the case? In the following article we compare three 1-Step probe-based RT-qPCR systems for amplification of viral RNA. We examine two viral RNA targets in singleplex and also evaluate multiplex performance with simultaneous detection of both viruses. These experiments show that an all-purpose master mix from Promega, GoTaq® Probe 1-Step RT-qPCR System, performs as well as products from QIAGEN and Life Technologies specifically named for use with pathogens or viruses.
Materials and Methods
We tested GoTaq® Probe 1-Step RT-qPCR System (Cat.# A6120) against two competitor products advertised to be optimized for virus and pathogen detection, AgPath-ID™ One-Step RT-PCR Reagents from Life Technologies and QuantiFast® Pathogen RT-PCR Kit from QIAGEN. To test each master mix for performance with viral amplification, a four-fold serial dilution was performed from 50,000 copies to 12 copies with RNA from HIV and Zika viruses (ATCC Cat.# VR-3245SD and VR-3252SD). Both viral RNAs were amplified in singleplex and multiplex reactions 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 with sequences listed below. Reactions were performed on an Applied Biosystems® 7500 Fast Real-Time PCR System.
Zika Primer/Probe Set Sequence¹:
Forward: CCG CTG 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
HIV and Zika RNA samples were amplified using GoTaq®, AgPath-ID™, and QuantiFast® Pathogen in singleplex (Figure 1) and as multiplexed reactions. Shown in Figure 2 is the single versus multiplex standard curves for Zika RNA for GoTaq® Probe 1-Step RT-qPCR System and AgPath-ID™. Both master mixes showed no significant differences in Cq values at all dilutions with RNA amplified in multiplex or singleplex. GoTaq® Probe 1-Step RT-qPCR System produced earlier Cq values with each dilution of viral RNA tested compared to AgPath-ID®, indicating more sensitive viral target amplification. QuantiFast® Pathogen exhibited significant Cq differences between singleplex and multiplex reactions for Zika RNA (data not shown). In Table 1, linearity and efficiencies for single and multiplex reactions are listed. The GoTaq® Probe 1-Step RT-qPCR System had excellent performance: R² values above 0.99 and efficiencies in the desiered range of 95-110%. Both competitor products showed similar linearity and efficiencies as the GoTaq® Probe 1-Step RT-qPCR System with the exception of AgPath-ID™, which had a 91% amplification efficiency for Zika RNA multiplex. These results suggest that the GoTaq® Probe 1-Step RT-qPCR System can perform equivalently or better than the leading master mixes that are labelled for use with pathogens.
Figure 1. Amplification of viral RNA with different master mixes. Serially diluted control RNA from HIV (left) or Zika (right) was amplified using GoTaq® Probe 1-Step RT-qPCR System, AgPath-ID™, or QuantiFast® Pathogen using manufacturers' recommended cycling conditions. Results are shown as the average ± standard deviation of four amplification reactions.
Figure 2. Sensitivity with singleplex or multiplex reactions. Zika virus RNA was amplified in single or multiplex with HIV RNA using either GoTaq® Probe 1-Step RT-qPCR System (left) or AgPath-ID™ (right).
Table 1. Amplification efficiency and linearity of viral RNA amplification in single and multiplex. Serially diluted viral RNA standards were amplified in single or multiplex using GoTaq® Probe 1-Step RT-qPCR System or competitor kits. Linearity (R²) and amplification efficiency (%) were determined from standard curves using the Applied Biosystems® 7500 software.
The GoTaq® Probe 1-Step RT-qPCR System is a flexible amplification system for detection of a diverse range of sample type RNAs, including viruses. Even though it is not directly marketed for viruses or pathogens, it amplifies viral RNA with equivalent or better sensitivity than specifically branded systems. The GoTaq® Probe 1-Step RT-qPCR System can also be used for the detection of multiple targets in a single reaction with no loss in amplification efficiency or linearity. The leading competitors use the word "pathogen" in their name, but GoTaq® Probe 1-Step RT-qPCR System is a sensitive, robust alternative for one-step pathogen detection.
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.
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