qScript XLT 1-Step RT-qPCR ToughMix

Tough-tested 1-step real time PCR

Features & Benefits

  • Tough Tested - Overcomes common inhibitors including polysacharides, heme/hemoglobin, humic acid, melanin
  • Flexible - Use fast or standard qPCR cycling conditions
  • Broad dynamic range- Reliable data from your precious samples every time
  • Multiplexing enabled - Supports highly sensitive detection for up to four targets

 

qScript XLT 1-Step RT-qPCR ToughMix is intended for molecular biology applications. This product is not intended for the diagnosis, prevention or treatment of a disease.

Description

qScript XLT One-Step RT-qPCR ToughMix is a ready-to-use, highly sensitive master mix for reverse transcription quantitative PCR (RT-qPCR) of RNA templates using hybridization probe detection chemistries such as TaqMan® 5'-hydrolysis probes. First-strand cDNA synthesis and subsequent PCR amplification are carried out seamlessly in the same reaction mixture with optimized 1-step thermal cycling parameters. This kit is ideal for highly sensitive quantification of RNA viruses or low abundance RNA targets as well as high throughput gene-expression studies. The system has been optimized to deliver maximum RT-PCR efficiency, sensitivity, and specificity in minimal reaction volumes and accelerated thermal cycling rates. The only necessary user-supplied materials for RT-qPCR is RNA sample and probe assay. It is compatible with all types of molecular probe assays including dual-labeling strategies. Elevating cDNA synthesis reaction temperature to 50-55°C during one-step RT-qPCR improves primer annealing and disruption of RNA secondary structure that can interfere with cDNA synthesis.
Featured Publication

High-throughput Detection of Respiratory Pathogens in Animal Specimens by Nanoscale PCR

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Details

  • Contents

    2X concentrated One-Step Master Mix containing:

    • Reaction buffer with optimized concentrations of molecular-grade MgCl2, dATP, dCTP, dGTP, dTTP
    • qScript XLT reverse transcriptase
    • RNase inhibitor protein
    • AccuStart II Taq DNA Polymerase
    • Inert AccuVue™ plate loading dye
    • Proprietary enzyme stabilizers and performance-enhancing additives
    • Titrated reference dye (if applicable)
  • Storage & Handling
    qScript XLT One-Step RT-qPCR ToughMix is stable for up to one year when stored in a constant temperature freezer at -20°C protected from light. Repeated freezing and thawing does not affect RT-qPCR performance.
  • 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
    Safety Data Sheets (SDS)
    CofA (PSF)
    Technical Notes
    Publications
    High Prevalence of Highly Variable Atypical Porcine Pestiviruses Found in Germany
    M. Beer, Transboundary and Emerging Diseases - 2016
    Abstract
    Recently, a novel atypical porcine pestivirus (APPV) with significant distribution was described in the USA. Subsequent screening of the German pig sector showed a high prevalence of APPV with high variability among strains. First indication of a cell culture isolate is provided which will allow further investigations like pathogenesis studies.
    Replication of human norovirus RNA in mammalian cells reveals a lack of interferon response
    Lin Qu, Journal of Virology - 2016
    Abstract
    Human noroviruses (HuNoVs), named after the prototype strain Norwalk virus (NV), are a leading cause of acute gastroenteritis outbreaks worldwide. Studies on the related murine norovirus (MNV) have demonstrated the importance of an interferon (IFN) response in host control of virus replication, but this remains unclear for HuNoVs. Despite the lack of an efficient cell culture infection system, transfection of stool-isolated NV RNA into mammalian cells leads to viral RNA replication and virus production. Using this system, we show here that NV RNA replication is sensitive to type I (α/β) and III (IL-29) IFN treatment. However, in cells capable of robust IFN response to Sendai virus (SeV) and poly(I:C), NV RNA replicates efficiently and generates double-stranded RNA without inducing a detectable IFN response. Replication of HuNoV genogroup GII.3 strain U201 RNA, generated from a reverse genetics system, also does not induce an IFN response. Consistent with a lack of IFN induction, NV RNA replication is neither enhanced by neutralization of type I/III IFNs through neutralizing antibodies or the soluble IFN decoy receptor B18R, nor by shRNA knockdown of MAVS or IRF3 in the IFN induction pathways. In contrast to other positive-strand RNA viruses that block IFN induction by targeting MAVS for degradation, MAVS is not degraded in NV RNA-replicating cells and a SeV-induced IFN response is not blocked. Together, these results indicate that HuNoV RNA replication in mammalian cells does not induce IFN response, suggesting that the IFN response may play a limited role in host restriction of HuNoV replication. IMPORTANCE Human noroviruses (HuNoVs) are a leading cause of epidemic gastroenteritis worldwide. Due to lack of an efficient cell culture system and robust small animal model, little is known about the innate host defense to these viruses. Studies on murine norovirus (MNV) have shown the importance of an interferon (IFN) response in host control of MNV replication, but this remains unclear for HuNoVs. Here we investigated the IFN response to HuNoV RNA replication in mammalian cells using Norwalk virus stool RNA transfection, a reverse genetics system, IFN neutralization reagents, and shRNA knockdown methods. Our results show that HuNoV RNA replication in mammalian cells does not induce an IFN response, nor can it be enhanced by blocking the IFN response. These results suggest a limited role of the IFN response in host control of HuNoV RNA replication, providing important insights into our understanding of host defense to HuNoVs that differs from MNV.
    Prevalence of influenza A virus in live-captured North Atlantic gray seals: a possible wild reservoir
    Wendy Blay Puryear, Emerging Microbes & Infections - 2016
    Abstract
    Influenza A virus (IAV) has been associated with multiple unusual mortality events (UMEs) in North Atlantic pinnipeds, frequently attributed to spillover of virus from wild-bird reservoirs. To determine if endemic infection persists outside of UMEs, we undertook a multiyear investigation of IAV in healthy, live-captured Northwest Atlantic gray seals (Halichoerus grypus). From 2013 to 2015, we sampled 345 pups and 57 adults from Cape Cod, MA, USA and Nova Scotia, Canada consistently detecting IAV infection across all groups. There was an overall viral prevalence of 9.0% (95% confidence interval (CI): 6.4%–12.5%) in weaned pups and 5.3% (CI: 1.2%–14.6%) in adults, with seroprevalences of 19.3% (CI: 15.0%–24.5%) and 50% (CI: 33.7%–66.4%), respectively. Positive sera showed a broad reactivity to diverse influenza subtypes. IAV status did not correlate with measures of animal health nor impact animal movement or foraging. This study demonstrated that Northwest Atlantic gray seals are both permissive to and tolerant of diverse IAV, possibly representing an endemically infected wild reservoir population.
    Toxic Accumulation of LPS Pathway Intermediates Underlies the Requirement of LpxH for Growth of Acinetobacter baumannii ATCC 19606
    Daryl L. Richie, PLOS ONE - 2016
    Abstract
    The lipid A moiety of lipopolysaccharide (LPS) is the main constituent of the outer leaflet of the Gram-negative bacterial outer membrane (OM) and is essential in many Gram-negative pathogens. An exception is Acinetobacter baumannii ATCC 19606, where mutants lacking enzymes occurring early in lipid A biosynthesis (LpxA, LpxC or LpxD), and correspondingly lacking LPS, can grow. In contrast, we show here that LpxH, an enzyme that occurs downstream of LpxD in the lipid A biosynthetic pathway, is essential for growth in this strain. Multiple attempts to disrupt lpxH on the genome were unsuccessful, and when LpxH expression was controlled by an isopropyl β- d -1-thiogalactopyranoside (IPTG) inducible promoter, cell growth under typical laboratory conditions required IPTG induction. Mass spectrometry analysis of cells shifted from LpxH-induced to uninduced (and whose growth was correspondingly slowing as LpxH was depleted) showed a large cellular accumulation of UDP-2,3-diacyl-GlcN (substrate of LpxH), a C14:0(3-OH) acyl variant of the LpxD substrate (UDP-3- O -[( R )-3-OH-C 14 ]-GlcN), and disaccharide 1-monophosphate (DSMP). Furthermore, the viable cell counts of the LpxH depleted cultures dropped modestly, and electron microscopy revealed clear defects at the cell (inner) membrane, suggesting lipid A intermediate accumulation was toxic. Consistent with this, blocking the synthesis of these intermediates by inhibition of the upstream LpxC enzyme using CHIR-090 abrogated the requirement for IPTG induction of LpxH. Taken together, these data indicate that LpxH is essential for growth in A . baumannii ATCC19606, because, unlike earlier pathway steps like LpxA or LpxC, blockage of LpxH causes accumulation of detergent-like pathway intermediates that prevents cell growth.
    Development of duplex dual-gene and DIVA real-time RT-PCR assays and use of feathers as a non-invasive sampling method
    Irit Davidson, Avian Pathology - 2016
    Abstract
    The avian flavivirus Turkey Meningoencephalitis Virus (TMEV) causes a neuroparalytic disease of commercial turkeys, expressed in paresis, incoordination, dropping wings and mortality that is controlled by vaccination. The molecular diagnosis using brain tissue RNA was now upgraded by the development of a diagnostic dual-gene multiplex real-time PCR targeting the env and the NS5 genes, increasing the sensitivity by 10-100 fold compared to the previously existing assays. Based on the recent complete sequences of 5 TMEV isolates we now developed a Differentiating Infected from Vaccinated Animals (DIVA) assay, to distinguish between wild-type TMEV strains and the vaccine virus. The DIVA was evaluated on commercial vaccines produced by two manufacturers, on RNA purified from brains of experimentally infected turkeys with TMEV strains, and on clinical samples collected between the years 2009-2015. We also investigated turkey feather pulps for their suitability to serve for TMEV detection, to avoid invasive sampling and bird killing. The parallel TMEV diagnosis in brain and feather-pulp RNA were similarly useful for diagnosis, at least, in experimentally-infected turkeys and in 3 cases of disease encountered in commercial flocks.
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