RapiDxFire qPCR 5X Master Mix GF

Formulated with diagnostic kit developers in mind, RapiDxFire qPCR 5X Master Mix glycerol free (GF) can be combined with gene-specific primers and hydrolysis probes for immediate use in high-throughput laboratory developed test (LDT) workflows or further processed for lyophilisation for use in point of care (POC) devices. Provided at a 5X concentration without passive reference dye, this flexible master mix allows more room for sample and up to 5 target detection at one time. The RapiDxFire qPCR 5X Master Mix, manufactured in an ISO 13485-certified facility, is available in small development sizes to large scale production batches to assist you through the diagnostic test development process.

RapiDxFire qPCR 5X Master Mix GF has been optimised for use with all Biosearch Technologies’ probes (Dual-Labelled BHQ, BHQplus, BHQnova and BHQplex CoPrimers), which all contain Biosearch Technologies’ proprietary BHQ quenchers. BHQ dyes have been proven to dramatically reduce low-level background fluorescence due to highly efficient static quenching between reporter and quencher. BHQ dyes are compatible with reporter fluorescent dyes that span the visible spectrum, allowing for broad flexibility in fluorophore selection. RapiDxFire qPCR 5X Master Mix GF can also be used with any probe-based assay. In addition, the Biosearch Technologies RealTimeDesign software is available online to facilitate the design of qPCR assays. Research use only, not for use in diagnostic procedures.

Applications

Pathogen detection
Food safety testing
Biomarker discovery and monitoring
Gene expression analysis in two-step RT-qPCR

Sensitive detection down to 10 DNA copies in singleplex and 5-plex qPCR assays

Figure 1. Singleplex (A) and multiplex (B) amplification plots with RapiDxFire qPCR Master Mix using 10-fold dilution series. cDNA was synthesized from MS2 phage RNA and amplified using five different assays with BHQ probes. The data displayed here is the target reported with Quasar 705. Template DNA (5 µL) was added to RapiDxFire qPCR Master Mix reaction mix (15 µL) and performed under standard cycling conditions. Sensitive and reproducible detection of 10 to 1,000,000 copies of cDNA was achieved. For both singleplex and multiplex reactions, standard curves for all targets generated a PCR efficiency of 90-110% and an R2 value of 0.995 to 0.999.

Automation-friendly 48 hour benchtop stability of fully assembled reactions

Figure 2. Amplification plots of a 4-plex RapiDxFire qPCR fully assembled reaction stored at room temperature over time prior to PCR cycling. We tested 1 hour, 24 hour or 48 hour pre-thermal cycling incubation times with reactions containing RapiDxFire qPCR Master Mix, BHQ assays, and DNA template. A multiplex assay targeting four individual mouse genomic DNA targets using BHQ probes and primers at a standard concentration of 200 nM and 900 nM, respectively, reporting with FAM and Biosearch Technologies proprietary dyes CAL Fluor Orange 560, CAL Fluor Red 610 and Quasar 670, was amplified using standard cycling conditions. A completely assembled reaction can be left on the bench for up to 48 hours without affecting the range of detection down to 10 copies.

Reliable lot to lot uniformity

Figure 3. The Cq mean and standard deviation of three independent lots of RapiDxFire qPCR Master Mix. Three lots of BHQ Probe Master Mix were each tested with BHQ probes addressing four individual targets in a multiplex reaction. The cumulative deviation was < Cq 0.5 between each replicate (3 x 10 replicates) at the 10 copy level across three different lots.

RapiDxFire qPCR Master Mix outperforms other master mixes in accurately quantifying pathogens during challenging multiplex reactions.

Sample Organism (1,000,000 copies/rxn) Organism (100 copies/rxn)
1 S. typhi P. aeruginosa & S. liquifacens & E. coli
2 E. coli P. aeruginosa & S. liquifacens & S. typhi
3 P. aeruginosa S. liquifacens & S. typhi & P. aeruginosa
4 S. liquifacens S. typhi & P. aeruginosa & S.typhi
5 S. typhi & E. coli P. aeruginosa & S. liquifacens
6 P. aeruginosa & S. liquifacens S. typhi & E. coli
7 P. aeruginosa & S. liquifacens & E. coli S. typhi

Figure 4. Comparison of RapiDxFire qPCR Master Mix to other competitor master mixes in quantifying Salmonella typhi among varying levels of other pathogenic bacteria. A series of multiplex experiments were performed to detect S. typhi in the background of increasingly abundant background of genomic DNA from pathogenic organisms, Escherichia coli, Pseudomonas aeruginosa, and Serratia liquefaciens. BHQ probe (200 nM) and primer (500 nM) concentrations were fixed for each experiment. Thermal cycling conditions were as stated by the manufacturer. Each reaction was run in 20 µL volumes with 5 µL of template to a final concentration of either 1,000,000 or 100 target copies (ratios indicated in Table 2). RapiDxFire qPCR Master Mix most accurately quantified S. typhi in all samples, and was the only master mix of appropriate sensitivity to reliably detect the presence of S. typhi in the most challenging sample

.

Key features

• 5X dye-free formulation offering flexible reaction setups and protocols
• Sensitive detection
• Wide dynamic range for multiplexing
• 48-hour reaction benchtop stability ideal for automated workflows
• Glycerol-free, Triton-free, high concentration, and bulk formulations for adaptable test development and lyophilisation options.
• Manufactured in an ISO 13485-certified facility demonstrating batch to batch reproducibility
• Component of the RapiDxFire 1-step RT-qPCR system for effective detection of viral RNA pathogens

Specifications

Concentration: 5X Master Mix

Storage: Store at -20 °C.

Master Mix buffer: Glycerol-free and Triton™ X-100-free buffer

Contaminating activity assays: RapiDxFire qPCR 5X Master Mix GF is free of detectable contaminating DNA exonuclease, and DNA endonuclease.

Product Details

RapiDxFire qPCR 5X Master Mix GF is also a component of the RapiDxFire 1-step RT-qPCR System.

For Laboratory Use.

Resources

Application notes

RapiDxFire qPCR5 X MasterMix GF

rapidxfire 1 step rt qpcr sars cov 2 detection

Documents

• 30050 1 RapiDxFire qPCR 5X Master Mix Glycerol Free 1 mL
• 30050 100ML RapiDxFire qPCR 5X Master Mix Glycerol Free 100 mL
• 30050 2 RapiDxFire qPCR 5X Master Mix Glycerol Free 10 mL
• quick guide rapidxfire qpcr 5x master mix gf
• user guide rapidxfire qpcr 5x master mix gf
• RapiDxFire qPCR5 X MasterMix GF
• rapidxfire 1 step rt qpcr sars cov 2 detection