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.

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
    Store components in a constant temperature freezer at -25°C to -15°C protected from light upon receipt. Repeated freezing and thawing does not impair product performance. For lot specific expiry date, refer to package label, Certificate of Analysis or Product Specification Form.ght.
  • 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
    • Quantabio Q
    • BioRad CFX
    • Roche LightCycler 480
    • Other
    Bio-Rad iCycler iQ systems
    • BioRad iCycler iQ™
    • BioRad MyiQ™
    • BioRad iQ™5
  • Related Resources
    Product Manuals
    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.
    Prevalence and associated risk factors of Giardia duodenalis infection among school-going children in Nepal
    Sarmila Tandukar, Parasitology Research - 2018
    Abstract
    This study aimed to determine the prevalence of intestinal parasites and its associated risk factors among school-going children in Kathmandu, Nepal. Between August and September 2016, a total of 333 stool samples were collected from children at five public schools. The collected samples were subjected to formol-ether concentration, followed by conventional microscopic examination for intestinal parasites. The overall prevalence of intestinal parasites was 24.3% (81/333), with Giardia spp. showing the highest prevalence of 18.9% (63/333). Samples positive for Giardia spp. by microscopy were further subjected to quantitative polymerase chain reaction (qPCR) for G. duodenalis, resulting in a positive ratio of 100%. The positive ratio of Giardia spp. was considerably high among children consuming tanker water (27.3%), jar water (21.0%), and tap water (17.5%). Our results demonstrated that G. duodenalis remains predominant in school-going children in Nepal.
    Strain-specific quantification of root colonization by plant growth promoting rhizobacteria Bacillus firmus I-1582 and Bacillus amyloliquefaciens QST713 in non-sterile soil and field conditions
    Hajeewaka C. Mendis, PLOS ONE - 2018
    Abstract
    Bacillus amyloliquefaciens QST713 and B. firmus I-1582 are bacterial strains which are used as active ingredients of commercially-available soil application and seed treatment products Serenade® and VOTiVO®, respectively. These bacteria colonize plant roots promoting plant growth and offering protection against pathogens/pests. The objective of this study was to develop a qPCR protocol to quantitate the dynamics of root colonization by these two strains under field conditions. Primers and TaqMan® probes were designed based on genome comparisons of the two strains with publicly-available and unpublished bacterial genomes of the same species. An optimized qPCR protocol was developed to quantify bacterial colonization of corn roots after seed treatment. Treated corn seeds were planted in non-sterile soil in the greenhouse and grown for 28 days. Specific detection of bacteria was quantified weekly, and showed stable colonization between ~104–105 CFU/g during the experimental period for both bacteria, and the protocol detected as low as 103 CFU/g bacteria on roots. In a separate experiment, streptomycin-resistant QST713 and rifampicin-resistant I-1582 strains were used to compare dilution-plating on TSA with the newly developed qPCR method. Results also indicated that the presence of natural microflora and another inoculated strain does not affect root colonization of either one of these strains. The same qPCR protocol was used to quantitate root colonization by QST713 and I-1582 in two corn and two soybean varieties grown in the field. Both bacteria were quantitated up to two weeks after seeds were planted in the field and there were no significant differences in root colonization in either bacteria strain among varieties. Results presented here confirm that the developed qPCR protocol can be successfully used to understand dynamics of root colonization by these bacteria in plants growing in growth chamber, greenhouse and the field.
    Assessment of Erysiphe necator ascospore release models for use in the Mediterranean climate of Western Oregon
    Lindsey D Thiessen, Plant Disease - 2018
    Abstract
    Predictive models have been developed in several major grape-growing regions to correlate environmental conditions to Erysiphe necator ascospore release; however, these models may not be broadly applicable in regions with different climatic conditions. To assess ascospore release in near-coastal regions of western Oregon, chasmothecia (syn. cleistothecia) were collected prior to leaf drop and placed onto natural and artificial grape trunk segments and overwintered outside. Ascospore release was monitored for three overwintering seasons using custom impaction spore traps from leaf drop (BBCH 97) until the onset of the disease epidemic in the following growing season. Airborne inoculum was concurrently monitored in a naturally infested research vineyard. Weather and ascospore release data were used to assess previously-developed models, and correlate environmental conditions to ascospore release. Ascospore release was predicted by all models prior to bud break (BBCH 08), and was observed from the first rain event following the start of inoculum monitoring until monitoring ceased. Previously developed models over-predicted ascospore release in the Willamette Valley and predicted exhaustion of inoculum prior to bud break. The magnitude of ascospore release could not be correlated to environmental conditions, thus a binary ascospore release model was developed where release is a function of the collective occurrence of the following factors within a 24-hour period: > 6 hours of cumulative leaf wetness during temperatures > 4 °C, precipitation > 2.5 mm, and relative humidity > 80%. The Oregon Model was validated using field collected ascospore datasets, and predicted ascospore release with 66% accuracy (P = 0.02). Extant methods for estimating ascospore release may not be sufficiently accurate to use as predictive models in wet, temperate climatic regions.
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