PerfeCTa Multiplex qPCR ToughMix

Advanced 1-tube SuperMix optimized to support highly multiplexed DNA amplifications in miniaturized reaction volumes and withstand a broad spectrum of PCR inhibitors

Features & Benefits

  • Tough tested - overcomes common qPCR inhibitors
  • Supports sensitive, highly-multiplex detection assays (5-target)
  • Supports fast or conventional PCR cycling
  • Broad linear dynamic range

 

PerfeCTa Multiplex qPCR ToughMix is intended for molecular biology applications. This product is not intended for the diagnosis, prevention or treatment of a disease.

Description

PerfeCTa MultiPlex qPCR ToughMix is a 5X concentrated, ready-to-use reaction cocktail for real-time quantitative PCR (qPCR) with ToughMix reagent technology that neutralizes a broad spectrum of PCR inhibitors that compromise assay performance with crude extracts, clinical specimens, plant, soil environmental or complex food matrix. The only necessary user-supplied materials are probe assays and DNA samples. Extra-concentrated reagent provide more flexibility with dilute DNA samples sensitivity. PerfeCta MultiPlex qPCR ToughMix has been rigorously optimized to deliver maximum assay precision, sensitivity and PCR efficiency in miniaturized reaction volumes with either conventional or accelerated thermal cycling conditions.

Suppression of low copy amplicons by high copy reference targets during multiplex co-amplification skews the apparent representation and quantification of low copy target sequences. PerfeCTa MultiPlex qPCR ToughMix transcends these limitations by enabling 6 orders of magnitude in sensitive, linear assay performance with concurrent amplification of four abundant targets at 106 each. PerfeCTa MultiPlex qPCR ToughMix results in multiplexed qPCR with dynamic range and sensitivity that are comparable to single-plex qPCR probe assays without the need to rigorously titrate primer concentration.

A key component of PerfeCTa® MultiPlex qPCR ToughMix® is an ultra pure, highly processive thermostable DNA polymerase that is combined with high avidity monoclonal antibodies. This proprietary polymerase mix is highly resistant to PCR inhibitors and provides an extremely stringent automatic hot-start allowing reaction assembly, and temporary storage, at room temperature prior to PCR amplification.

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Details

  • Contents

    Single-tube, 5X concentrated reagent containing:

    • Reaction buffer with optimized concentrations of molecular-grade MgCl2, dATP, dCTP, dGTP, dTTP.
    • AccuStart II Taq DNA Polymerase.
    • Proprietary enzyme stabilizers and performance-enhancing additives.
    • Titrated reference dye (if applicable).
  • Storage & Handling
    PerfeCTa® Multiplex qPCR ToughMix® is stable for up to 2 years when stored in a constant temperature freezer at -20°C, protected from light. Repeated freezing and thawing of the reagent will not affect assay performance. Reagent may also be stored unfrozen at 4°C for up to 6 months, protected from light.
  • Instrument Capability
    ROX
    • Applied Biosystems 5700
    • Applied Biosystems 7000
    • Applied Biosystems 7300
    • Applied Biosystems 7700
    • Applied Biosystems 7900
    • Applied Biosystems 7900HT
    • Applied Biosystems 7900 HT Fast
    • Applied Biosystems StepOne™
    • Applied Biosystems StepOnePlus™
    Low ROX
    • Applied Biosystems 7500
    • Applied Biosystems 7500 Fast
    • Stratagene Mx3000P®
    • Stratagene Mx3005P™
    • Stratagene Mx4000™
    • Applied Biosystems ViiA 7
    • Applied Biosystems QuantStudio™
    • Agilent AriaMx
    • Douglas Scientific IntelliQube®
    • QIAGEN Rotor-Gene Q
    No ROX
    • BioRad CFX
    • Roche LightCycler 480
    • Other
    Bio-Rad iCycler iQ systems
    • BioRad iCycler iQ™
    • BioRad MyiQ™
    • BioRad iQ™5
  • Related Resources
    Product Manuals
    Product Flyers
    Safety Data Sheets (SDS)
    CofA (PSF)
    Publications
    Detection and Quantification of Acute Myeloid Leukemia-Associated Fusion Transcripts
    Jonathan Schumacher, Acute Myeloid Leukemia - 2017
    Abstract
    Real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR)-based detection of abnormal fusion transcripts is an important strategy for the diagnosis and monitoring of patients with acute myeloid leukemia (AML) with t(8;21)(q22;q22); RUNX1-RUNX1T1, inv(16)(p13.1;q22); CBFB-MYH11 or t(15;17)(q22;q12); PML-RARA. In RT-qPCR assays, patient-derived cDNA is subjected to amplification using PCR primers directed against the fusion transcript of interest as well as a reference gene for normalization. Quantification is typically performed by constructing standard curves for each PCR run using a series of plasmid standards of known concentration that harbor the same fusion transcript or the same reference gene of interest. Fusion transcripts and reference gene copy numbers are then calculated in patient samples using these standard curves. The process of constructing standard curves is laborious and consumes additional reagents. In this chapter, we give the method details for a multiplex RT-qPCR strategy to detect and quantify the acute myeloid leukemia (AML)-associated fusion transcripts PML-RARA in patients with t(15;17) without the need for standard curves. This general method can also be applied to other AML-associated fusion transcripts such as CBFB-MYH11 and RUNX1-RUNX1T1.
    Crystal digital droplet PCR for detection and quantification of circulating EGFR sensitizing and resistance mutations in advanced non-small cell lung cancer
    Cécile Jovelet, PLOS ONE - 2017
    Abstract
    Over the past years, targeted therapies using tyrosine kinase inhibitors (TKI) have led to an increase in progression-free survival and response rate for a subgroup of non-small cell lung cancer (NSCLC) patients harbouring specific gene abnormalities compared with chemotherapy. However long-lasting tumor regression is rarely achieved, due to the development of resistant tumoral subclones, which requires alternative therapeutic approaches. Molecular profile at progressive disease is a challenge for making adaptive treatment decisions. The aim of this study was to monitor EGFR-mutant tumors over time based on the quantity of mutant DNA circulating in plasma (ctDNA), comparing two different methods, Crystal™ Digital™ PCR and Massive Parallel Sequencing (MPS). In plasma circulating cell free DNA (cfDNA) of 61 advanced NSCLC patients we found an overall correlation of 78% between mutated allelic fraction measured by Crystal Digital PCR and MPS. 7 additional samples with sensitizing mutations and 4 additional samples with the resistance mutation were detected with Crystal Digital PCR, but not with MPS. Monitoring levels of both mutation types over time showed a correlation between levels and trends of mutated ctDNA detected and clinical assessment of disease for the 6 patients tested. In conclusion, Crystal Digital PCR exhibited good performance for monitoring mutational status in plasma cfDNA, and also appeared as better suited to the detection of known mutations than MPS in terms of features such as time to results.
    Calcium transcriptionally regulates the biofilm machinery of Xylella fastidiosa to promote continued biofilm development in batch cultures
    Jennifer Parker, Environmental Microbiology - 2016
    Abstract
    The functions of calcium (Ca) in bacteria are less characterized than in eukaryotes, where its role has been studied extensively. The plant-pathogenic bacterium Xylella fastidiosa has several virulence features that are enhanced by increased Ca concentrations, including biofilm formation. However, the specific mechanisms driving modulation of this feature are unclear. Characterization of biofilm formation over time showed that 4 mM Ca supplementation produced denser biofilms that were still developing at 96 h, while biofilm in non-supplemented media had reached the dispersal stage by 72 h. To identify changes in global gene expression in X. fastidiosa grown in supplemental Ca, RNA-Seq of batch culture biofilm cells was conducted at three 24-h time intervals. Results indicate that a variety of genes are differentially expressed in response to Ca, including genes related to attachment, motility, exopolysaccharide synthesis, biofilm formation, peptidoglycan synthesis, regulatory functions, iron homeostasis, and phages. Collectively, results demonstrate that Ca supplementation induces a transcriptional response that promotes continued biofilm development, while biofilm cells in nonsupplemented media are driven towards dispersion of cells from the biofilm structure. These results have important implications for disease progression in planta, where xylem sap is the source of Ca and other nutrients for X. fastidiosa.
    Calcium transcriptionally regulates the biofilm machinery of Xylella fastidiosa to promote continued biofilm development in batch cultures
    Jennifer Parker, Environmental Microbiology - 2016
    Abstract
    The functions of calcium (Ca) in bacteria are less characterized than in eukaryotes, where its role has been studied extensively. The plant-pathogenic bacterium Xylella fastidiosa has several virulence features that are enhanced by increased Ca concentrations, including biofilm formation. However, the specific mechanisms driving modulation of this feature are unclear. Characterization of biofilm formation over time showed that 4 mM Ca supplementation produced denser biofilms that were still developing at 96 h, while biofilm in non-supplemented media had reached the dispersal stage by 72 h. To identify changes in global gene expression in X. fastidiosa grown in supplemental Ca, RNA-Seq of batch culture biofilm cells was conducted at three 24-h time intervals. Results indicate that a variety of genes are differentially expressed in response to Ca, including genes related to attachment, motility, exopolysaccharide synthesis, biofilm formation, peptidoglycan synthesis, regulatory functions, iron homeostasis, and phages. Collectively, results demonstrate that Ca supplementation induces a transcriptional response that promotes continued biofilm development, while biofilm cells in nonsupplemented media are driven towards dispersion of cells from the biofilm structure. These results have important implications for disease progression in planta, where xylem sap is the source of Ca and other nutrients for X. fastidiosa.
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