Boost Microbial Detection Using Optimized 1-Step RT-qPCR Reagents

TaqPath DuraPlex 1-Step RT-qPCR Master Mix Enable automation with benchtop-stable reagents qPCR reagents Product overview The Applied Biosystems™ TaqPath™ DuraPlex™ 1-Step RT-qPCR Master Mix is an automation-ready, single-tube master mix optimized for detection of viral and bacterial pathogens even in the presence of PCR inhibitors. The 4X concentrated formulation is capable of multiplexing 6 targets in a single well with exceptional sensitivity and specificity. This fast-cycling general purpose reagent (GPR) is ideal for clinical, biopharma, and research customers who need a rapid, sensitive, and reproducible master mix for testing and developing high-throughput 1-step RT-qPCR assays. We offer the TaqPath DuraPlex 1-Step RT-qPCR Master Mix in 4X concentration, with two formulations: • TaqPath DuraPlex 1-Step RT-qPCR Master Mix • TaqPath DuraPlex 1-Step RT-qPCR Master Mix (No ROX™) The benefits of TaqPath DuraPlex 1-Step RT-qPCR Master Mix help you: • Automate your workflow with benchtop stability of completely assembled reactions at room temperature for a full workday • Optimize your assay for high-order multiplexing of up to 6 targets in a single reaction • Improve handling, in-use stability, and freeze-thaw conditions • Increase confidence in detecting both RNA and DNA targets in samples containing challenging PCR inhibitors • Ensure reproducible results with consistent lot-to-lot performance Automates your workflow TaqPath DuraPlex 1-Step RT-qPCR Master Mix provides a full work day (over 8 hours) of stability of completely assembled reactions (master mix, sample, and assay) to enable the use of liquid handling and other automation equipment. This fast-cycling master mix can be used effectively for both RNA and DNA detection of bacterial, viral, and fungal targets. Figure 1 below shows consistent sensitivity with a multiplex assay tested at 0 and 8 hours compared to other manufacturers. Target Master mix TaqPath DuraPlex ROX Bio-Rad Reliance Qiagen QuantiNova dCq 3 2 1 0 –1 –2 –3 4 –4 –5 –6 FZDI FN1 RNase P B2M TFRC FZDI FN1 RNase P B2M TFRC FZDI FN1 RNase P B2M TFRC Master mix dCq 3 2 0 –1 –2 –3 Target ACADM HPRT1 ABCC2 SPG7 FBN1 ACADM HPRT1 ABCC2 SPG7 FBN1 ACADM HPRT1 ABCC2 SPG7 FBN1 TaqPath DuraPlex, No ROX Quantabio UltraPlex Roche LightCycler Optimizes high-order multiplexing TaqPath DuraPlex 1-Step RT-qPCR Master Mix has been validated with multiplex assays to detect up to 6 targets in a single well with the formulation without ROX dye and up to 5 targets in a single well with the ROX formulation. TaqPath DuraPlex 1-Step RT-qPCR Master Mix enables accurate detection of both low and high concentrations of RNA and DNA in samples and provides a wide dynamic range. Figure 2 demonstrates the excellent PCR linearity over an input range of 8 logarithmic units in a 5-plex assay. Figure 1. TaqPath DuraPlex 1-Step RT-qPCR Master Mix shows less change in Cq after 8 hours at room temperature (24°C), in assembled reactions, compared to master mixes from other suppliers. Assembled reactions consisted of master mix, RNA, and a 5-plex human gene expression assay. The shaded region on each graph denotes ±1 Cq change from comparison of a reaction at 8 hours and a reaction at 0 hour (control). (A) Master mixes used: blue = TaqPath DuraPlex 1-Step RT-qPCR Master Mix (No ROX); orange = Quantabio™ UltraPlex™ 1-Step ToughMix™ master mix; and green = Roche™ LightCycler™ Multiplex RNA Virus Master Mix. (B) Master mixes used: blue = TaqPath DuraPlex 1-Step RT-qPCR Master Mix (with ROX passive reference dye); orange = Bio-Rad™ Reliance One-Step Multiplex RT-qPCR Supermix; and green = Qiagen™ QuantiNova™ Multiplex RT-PCR Kit. Exceptional dynamic range of TaqPath DuraPlex 1-Step RT-qPCR Master Mix ∆Rn Cycle ∆Rn 5 10 15 20 25 30 35 40 5 10 15 20 25 30 35 40 Cycle FAM dye 1 x 107 1 x 106 1 x 105 1 x 104 1 x 103 1 x 107 1 x 106 1 x 105 1 x 104 1 x 103 VIC dye Amplification plot Amplification plot 5 10 15 20 25 30 35 40 5 10 15 20 25 30 35 40 Amplification plot Amplification plot Cycle Cycle JUN dye 1 x 107 1 x 106 1 x 105 1 x 104 1 x 103 1 x 107 1 x 106 1 x 105 1 x 104 1 x 103 Cy5 dye ∆Rn ∆Rn ∆Rn 5 10 15 20 25 30 35 40 Amplification plot Cycle ABY dye 1 x 107 1 x 106 1 x 105 1 x 104 1 x 103 Figure 2. Amplification plots from a 5-plex multiplex RT-qPCR reaction using TaqPath DuraPlex 1-Step RT-qPCR Master Mix demonstrate a wide input of 8-log dilution series of a synthetic RNA control for all 5 targets tested. R2 values are 0.99–1.0 and PCR efficiency is 99–103% for all targets. RNA input starts from 2.5 ng/20 µL reaction to 0.025 fg/20 µL reaction. A B Assembled reaction stability with TaqPath DuraPlex 1-Step RT-qPCR Master Mix 2 qPCR reagents thermofisher.com/duraplex Improves handling TaqPath DuraPlex 1-Step RT-qPCR Master Mix has been formulated to improve in-use handling with a lower viscosity to allow for more effective pipetting, including use with automated liquid-handling platforms. This master mix is stable at room temperature (24°C) for 1 week and can be stored under refrigeration (4°C) for a month after first use. The TaqPath DuraPlex 1-Step RT-qPCR Master Mix can be used confidently up to 8 freeze-thaw cycles without loss of performance. The novel formulation helps ensure that the master mix can be adapted to a variety of workflow scenarios. Figure 3 shows consistent performance of reagents stored for 1 week at room temperature. Storage temperature –20°C 24°C Cq Cycle limit Dilution (D) D1 D2 D3 D4 D5 D6 NTC D1 D2 D3 D4 D5 D6 NTC D1 D2 D3 D4 D5 D6 NTC D1 D2 D3 D4 D5 D6 NTC D1 D2 D3 D4 D5 D6 NTC Reporter dye FAM VIC ABY Cyanine 5 Cyanine 5.5 30 20 10 0 Figure 3. There is no change in performance of TaqPath DuraPlex 1-Step RT-qPCR Master Mix after storage for 1 week at 24°C, compared to product stored at –20°C. After 1 week of storage, a dilution series of RNA input was tested in a 5-plex multiplex reaction with TaqPath DuraPlex 1-Step RT-qPCR Master Mix. Blue bars indicated Cq values of control master mix stored at –20°C. Orange bars indicate Cq values after the master mix has been stored for 1 week at 24ºC. Each error bar is constructed using 1 standard deviation from the mean. dCq 16 14 12 10 8 6 4 2 0 50% viral transport medium 0.002 U/µL heparin 40 µM hematin inhibitor TaqPath DuraPlex (No ROX) TaqPath DuraPlex BioRad Reliance Quantabio UltraPlex Roche LightCycler Qiagen QuantiNova Figure 4. TaqPath DuraPlex 1-Step RT-qPCR Master Mix shows the least amount of Cq shift when challenged with three potential inhibitors of 1-step qPCR, compared to other 1-step master mixes. Viral transport medium was tested at 50% of total reaction volume, heparin was tested at 0.002 U/µL reaction, and hematin was tested at a concentration of 40 µM in the final reaction. TaqPath DuraPlex 1-Step master mix stability after 1 week at 24°C Increases confidence Superior inhibitor tolerance of TaqPath DuraPlex 1-Step RT-qPCR Master Mix The unique proprietary formulation of the TaqPath DuraPlex 1-Step RT-qPCR Master Mix allows robust performance even in the presence of substances that can inhibit PCR. This feature results in effective template detection from both purified RNA and DNA, including those isolated from challenging clinical sample matrices such as viral transport medium (VTM), stool, blood, and urine. Figure 4 shows TaqPath DuraPlex 1-Step RT-qPCR Master Mix performance with common PCR inhibitors compared to that of other manufacturers. The master mix was less affected by the presence of these inhibitors and RT-qPCR results were achieved with minimal loss of sensitivity, even at concentrations that inhibit other products tested. 3 qPCR reagents thermofisher.com/duraplex 30 25 20 15 10 5 0 Lot Cq Lot 1 Lot 2 Lot 3 Lot 1 Lot 2 Lot 3 Lot 1 Lot 2 Lot 3 Lot 1 Lot 2 Lot 3 Lot 1 Lot 2 Lot 3 Lot 1 Lot 2 Lot 3 B2M FN1 FZD1 GAPDH RNase P TFRC Target RNase P 30 25 20 15 10 5 0 Lot Cq Lot 1 Lot 2 Lot 3 Lot 1 Lot 2 Lot 3 Lot 1 Lot 2 Lot 3 Lot 1 Lot 2 Lot 3 Lot 1 Lot 2 Lot 3 B2M FN1 FZD1 RNase P RNase P TFRC Target Ordering information Description Quantity Cat. No. TaqPath DuraPlex 1-Step RT-qPCR Master Mix 0.5 mL A58666 5 x 1 mL A58667 10 mL A58668 TaqPath DuraPlex 1-Step RT-qPCR Master Mix (No ROX) 0.5 mL A58669 5 x 1 mL A58670 10 mL A58671 For Laboratory Use. © 2024 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. Quantabio, UltraPlex, and ToughMix are trademarks of Quantabio, LLC. Roche and LightCycler are trademarks of Roche Molecular Systems, Inc. Bio-Rad is a trademark of Bio-Rad Laboratories, Inc. Qiagen and QuantiNova are trademarks of Qiagen, GmbH. Cy is a registered trademark of Cytiva. COL121898 0124 Learn more at thermofisher.com/duraplex * No purchase necessary to receive an evaluation sample. Participation is open only to life science professionals in the US (excluding Puerto Rico) and Canada who are 21 years or older. Completed evaluation sample request forms must be received no later than December 31, 2024, at 6:00 p.m. PT, or until evaluation samples are depleted, whichever comes first. The sample size is 0.5 mL. Limit one evaluation sample per lab. By submitting your evaluation sample request response, you warrant that you are not prohibited by employment, contract, or law from accepting one evaluation sample from Life Technologies. Cannot be combined with other discounts or promotions. Offer void where prohibited, licensed, or restricted by federal, state, provincial, or local laws or regulation or agency/institutional policy. Other restrictions may apply. Helps ensure reproducibility This GPR is labeled “For Laboratory Use” and is manufactured in an ISO 13485–certified and FDA-registered facility, which adheres to current good manufacturing practice (CGMP) principles. All lots of TaqPath DuraPlex 1-Step RT-qPCR Master Mix are functionally tested to help ensure lot-to-lot reproducibility for Cq values and consistency in dynamic range across a wide variety of assays. Figure 5 shows consistent performance of 3 lots of TaqPath DuraPlex 1-Step RT-qPCR Master Mix in 6-plex and 5-plex assays. Compliance package A full suite of frequently requested quality compliance documents is available at thermofisher.com/duraplex. Figure 5. Three unique lots of TaqPath DuraPlex 1-Step RT-qPCR Master Mix were tested for consistency in performance. (A) Cq values for all 6 gene targets are similar in a multiplex reaction tested with the version of the product with no passive reference dye. (B) Cq values for all 5 gene targets are similar in a multiplex reaction tested with the ROX version of the product. A B Lot-to-lot consistency with TaqPath DuraPlex 1-Step RT-qPCR Master Mix Request a sample*