PerfeCTa qPCR ToughMix
Tough-tested real-time PCR
Features & Benefits
- ToughMix additives neutralize PCR inhibitors to ensure reliable assay performance with a spectrum of starting materials including: clinical specimens, plants, soil, environmental or complex food matrices.
- Superior assay sensitivity and target precision with AccuStart™ II enzyme technology – maximum-yielding, engineered Taq DNA polymerase with stringent, ultrapure antibody hotstart.
- Easy-to-use 2X concentrated master mixes with AccuVue™ plate loading dye and optimized passive reference dye for simplified reaction setup.
- Supports efficient vortex mixing with proprietary anti-foaming technology.
- Consistent and reliable performance with exceptional reagent stability (uninhibited after 20X freeze-thaw or 30 days at 22°C).
PerfeCTa qPCR ToughMix is intended for molecular biology applications. This product is not intended for the diagnosis, prevention or treatment of a disease.
PerfeCTa qPCR ToughMix is a 2X concentrated ready-to-use reaction cocktail for PCR amplification of DNA templates that relieves several types of PCR inhibition commonly encountered with crude extracts, environmental specimens, plant tissues, animal tissues, and complex food matrices. This robust real-time qPCR reagent provides maximum sensitivity and PCR efficiency with a variety of fluorogenic probe chemistries, including TaqMan® hydrolysis probes. The only user-supplied components are primers, probe(s), and DNA template. Pre-blended with inert AccuVue plate loading dye to help minimize pipette errors during setup and provides visual confirmation of thorough mixing. A key component of PerfeCTa 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. PerfeCTa qPCR ToughMix delivers exceptional performance with either fast or conventional PCR cycling protocols. UNG containing versions are blended with Uracil N-glycosylase to eliminate potential post-PCR carryover contamination associated with routine molecular testing.
Single-tube, 2X concentrated reagent containing:
- Reaction buffer with optimized concentrations of molecular-grade MgCl2, dATP, dCTP, dGTP, dTTP.
- AccuStart II Taq DNA Polymerase.
- Inert AccuVue dye.
- Proprietary enzyme stabilizers and performance-enhancing additives.
- Titrated reference dye (if applicable).
Storage & HandlingPerfeCTa qPCR ToughMix is stable for 2 years when stored in a constant temperature freezer at or below -20°C. For convenience, it may be stored unfrozen at 4°C for up to 6 months. After thawing, mix thoroughly before using. Repeated freezing and thawing does not affect PCR performance.
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Labor and consumable costs are major barriers for implementation of CDC recommendation of testing all stools for both O157 and non-O157. We evaluated the feasibility of a pooled nucleic acid amplification test (NAAT) as an approach for screening of stool specimens for STEC. For retrospective evaluation, 300 stool specimens were used to create pools of 10. The sensitivity was 83% for the pre-enrichment pooling strategy and 100% for the post-enrichment pooling strategy compared with individual NAAT. The difference in cycle threshold between individual and pooled NAAT results for specimens was significantly lower and more consistent for post-enrichment pooling (mean stx1 = 3.90, stx2 = 4.28) compared with pre-enrichment pooling (excluding undetected specimens, mean stx1 = 9.34, stx2 = 8.96) (P≤0.0013). Consumable cost and labor savings of 48-81% and 6-66%, respectively, were estimated for testing of 90 specimens by the post-enrichment pooled NAAT strategy based on an expected 1-2% positivity rate. A 30-day prospective, head-to-head clinical trial involving 512 specimens confirmed the sensitivity and labor savings associated with the post-enrichment pooled NAAT strategy. The post-enrichment pooled NAAT strategy described here is suitable for efficient, large-scale surveillance of all STEC serotypes. Comprehensive detection of STEC will result in accurate estimates of STEC burden and, consequently, appropriate public health interventions.Temperature modulates liver lipid accumulation in Atlantic salmon (Salmo salar L.) fed low dietary levels of long-chain n-3 fatty acidsN.h. Sissener, Aquaculture Nutrition - 2016AbstractAtlantic salmon (Salmo salar) were fed five graded levels of eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3), from 1.4 to 5.2% of total fatty acids (FA, 5–17 mg kg−1 feed), and grew from ~160 g to ~3000 g, with the period from 1450 g onwards conducted both at 6 °C and at 12 °C. All fish appeared healthy, and there were no diet-related differences in haematological or plasma parameters, as well as intestinal histological or gut microbiota analysis. Fish reared at 6 °C had higher accumulation of storage lipids in the liver compared to fish reared at 12 °C. Liver lipids also increased with decreasing dietary EPA + DHA at 6 °C, while there was no such relationship at 12 °C. Gene expression of SREBP1 and 2, LXR, FAS and CPT1 could not explain the differences in liver lipid accumulation. In liver polar lipids, DHA was found to be reduced when dietary EPA + DHA was <2.7% of FAs, while the level of EPA in the membranes was not affected. In conclusion, reducing dietary EPA + DHA from 5.2 to 1.4% of total FAs had a minor impact on fish health. Temperature was the factor that most affected the liver lipid accumulation, but there was also an interaction with dietary components.Mitochondrial DNA deletion percentage in sun exposed and non sun exposed skinJulia M. Powers, Journal of Photochemistry and Photobiology B: Biology - 2016AbstractThe percentages of mitochondrial genomes carrying the mtDNA3895 and the mtDNA4977 (common) deletion were quantified in sun exposed and non sun exposed skin biopsies, for five cohorts of patients varying either in sun exposure profile, age or skin cancer status. Non-melanoma skin cancer diagnoses are rising in Ireland and worldwide  but most risk prediction is based on subjective visual estimations of sun exposure history. A quantitative objective test for pre-neoplastic markers may result in better adherence to sun protective behaviours. Mitochondrial DNA (mtDNA) is known to be subject to the loss of a significant proportion of specific sections of genetic code due to exposure to ultraviolet light in sunlight. Although one such deletion has been deemed more sensitive, another, called the mtDNA4977 or common deletion, has proved to be a more useful indicator of possible risk in this study. Quantitative molecular analysis was carried out to determine the percentage of genomes carrying the deletion using non sun exposed and sun exposed skin biopsies in cohorts of patients with high or low sun exposure profiles and two high exposure groups undergoing treatment for NMSC. Results indicate that mtDNA deletions correlate to sun exposure; in groups with high sun exposure habits a significant increase in deletion number in exposed over non sun exposed skin occurred. An increase in deletion percentage was also seen in older cohorts compared to the younger group. The mtDNA3895 deletion was detected in small amounts in exposed skin of many patients, the mtDNA4977 common deletion, although present to some extent in non sun exposed skin, is suggested to be the more reliable and easily detected marker. In all cohorts except the younger group with relatively lower sun exposure, the mtDNA4977 deletion was more frequent in sun exposed skin samples compared to non-sun exposed skin.Three-color crystal digital PCRJ. Madic, Biomolecular Detection and Quantification - 2016AbstractDigital PCR is an exciting new field for molecular analysis, allowing unprecedented precision in the quantification of nucleic acids, as well as the fine discrimination of rare molecular events in complex samples. We here present a novel technology for digital PCR, Crystal Digital PCR™, which relies on the use of a single chip to partition samples into 2D droplet arrays, which are then subjected to thermal cycling and finally read using a three-color fluorescence scanning device. This novel technology thus allows three-color multiplexing, which entails a different approach to data analysis. In the present publication, we present this innovative workflow, which is both fast and user-friendly, and discuss associated data analysis issue, such as fluorescence spillover compensation and data representation. Lastly, we also present proof-of-concept of this three-color detection system, using a quadriplex assay for the detection of EGFR mutations L858R, L861Q and T790M.Rapid and accurate identification of Xanthomonas citri subspecies citri by fluorescence in situ hybridisationClick here to see all PublicationsDavid Waite, Letters in Applied Microbiology - 2016AbstractCitrus canker is an economically important disease caused by the bacterial pathogen Xanthomonas citri subsp. citri (Xcc). This organism targets a wide range of citrus plants, including sweet orange, grapefruit, lemon and lime. As Xcc is spread by environmental factors such as wind and rain, it is difficult to control its movement once the disease has established. In order to facilitate monitoring of citrus canker we sought to design a novel diagnostic protocol based on fluorescence in situ hybridisation (FISH) for identification of bacterial cells directly from canker pustules without cultivation or DNA extraction. This method was validated for specificity against a range of Xanthomonas species and strains. We show that our assay is extremely rapid (typically requiring between two and three hours), and possesses a similar specificity to existing PCR diagnostic tools. The sensitivity of the assay is comparable to that of an existing PCR-based technique and sufficient for identifying Xcc in symptomatic plant material. The method is easily transferable to diagnosticians without prior experience using FISH. This article is protected by copyright. All rights reserved.