Applications

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.

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.

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.

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

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