mericon L. monocytogenes Kit
For the detection of pathogens in food or animal feed samples using real-time PCR, Highly sensitive and specific detection of a broad range of pathogens, Streamlined and uniform detection protocol for all targets, Coupled to dedicated sample preparation solutions for all pathogen types, PCR run time of 73 minutes on QIAGEN's Rotor-Gene Q, Full license for PCR without additional costs
Each mericon PCR Assay is a multiplex PCR that amplifies both a specific DNA target and an internal control with high specificity. The internal control provides data regarding the presence of inhibitors in the tested samples and the overall success of the PCR. Each mericon PCR Assay includes a PCR primer set for a pathogen-specific target sequence, probes labeled with two distinct fluorescent dyes (FAM and MAX NHS Ester), positive control DNA, and all of the reagents necessary to perform the analysis. The Multiplex PCR Master Mix included in each kit contains QIAGEN proprietary technology, including HotStarTaq Plus DNA Polymerase, patented multiplex PCR technology such as Factor MP, and fast cycling technology including Q-bond. Multiplex PCR Master Mix is also highly tolerant to PCR inhibitors.
Features of the mericon Pathogen Detection Assays include:
- Streamlined protocol for pathogen detection that delivers results 3 hours after primary enrichment
- Exceptional sensitivity even with difficult food matrices and challenging pathogen strains
- Standardized detection protocols across all assays so a variety of targets can be analyzed in a single thermal cycler run
- Dedicated sample preparation solutions for an optimal overall workflow performance
- Innovative chemistry that is highly resistant to the inhibitors commonly found in complex foods
Pathogen detection by PCR is based on the amplification of a specific region of the relevant pathogen genome. In real-time PCR the amplified product is detected via target-specific fluorescent probes that bind to the amplified product. Accumulation of the PCR product results in an increased fluorescent signal from the bound probes. Monitoring the fluorescence intensities during the PCR run (i.e., in real time) allows the detection of the accumulating PCR product without having to re-open the reaction tubes afterward.
The probes in mericon PCR Assays are sequence-specific oligonucleotides with a fluorophore and a quencher moiety attached. The fluorophore is at the 5' end of the probe, and the quencher moiety is located at the 3' end. If the target DNA sequence is present, the probe is cleaved by the 5'–3' exonuclease activity of HotStarTaq Plus DNA Polymerase during the extension phase of PCR. This separates the fluorophore and the quencher moiety resulting in a detectable fluorescence that is proportional to the amount of accumulated PCR product.
The PCR primer set for each assay is highly specific and targets a unique and conserved DNA region of the tested pathogen genome that is verified bioinformatically and experimentally. Cross-reactivity has been thoroughly tested with a panel of selected targets for each mericon PCR Assay. Each assay can detect as few as 10 target DNA copies in a reaction.
Dedicated sample preparation solutions are available from QIAGEN for a broad range of starting materials. These solutions were developed to complement mericon PCR Assays, and provide a complete and efficient workflow for food safety testing.
HotStarTaq Plus DNA Polymerase
HotStarTaq Plus DNA Polymerase is a modified form of QIAGEN Taq DNA Polymerase. It is provided in an inactive state and has no enzymatic activity at ambient temperature, thereby preventing the formation of misprimed products and primer-dimers during reaction setup and the first denaturation step. Competition for reactants by PCR artifacts is therefore avoided, enabling high PCR specificity and accurate quantification. The enzyme is activated first at the start of a reaction by a 5-minute, 95°C incubation step, which enables reactions to be set up rapidly and conveniently at room temperature. In addition, the concentration of the polymerase in the master mix is optimized to allow short extension times.
Multiplex PCR Master Mix
The Multiplex PCR Master Mix was specifically developed for fast-cycling, multiplex, real-time PCR using sequence-specific probes. A novel additive in the buffer, Q-Bond, allows short cycling times on standard cyclers and on fast cyclers with rapid ramping rates. Q-Bond increases the affinity of HotStarTaq Plus DNA Polymerase for short single-stranded DNA, reducing the time required for primer/probe annealing to a few seconds. The buffer also contains Factor MP, which facilitates multiplex PCR. This synthetic factor increases the local concentration of primers and probes at the DNA template and stabilizes specifically bound primers and probes, allowing efficient annealing and extension. In addition, the Multiplex PCR Buffer is carefully formulated to be highly tolerant to the inhibitors commonly present in food.
QuantiTect Nucleic Acid Dilution Buffer
QuantiTect Nucleic Acid Dilution Buffer is an optimized solution to dilute the nucleic acids used as positive controls for mericon PCR Assays. The buffer stabilizes DNA controls during dilution and reaction setup and prevents loss of nucleic acids on plastic surfaces, such as tubes or pipet tips. The buffer is ready to use and is free of DNases. Proper use of the buffer enables safe and accurate dilution of the small amounts of nucleic acids typically used as controls for analysis of nucleic acids. Aliquots of diluted positive control can be stored in QuantiTect Nucleic Acid Dilution Buffer at -15 to -30°C for up to 6 months. Repeated freezing and thawing should be avoided.
ROX Dye Solution, 50x
ROX Dye Solution is used with certain real-time cyclers to compensate for non-PCR-related variations in fluorescence detection. It is provided in a 50x solution for your convenience. It is not needed for Rotor-Gene Q, LightCycler systems from Roche, SmartCycler instruments from Cepheid, and Bio-Rad instruments. It is necessary for most instruments from Applied Biosystems and is optional for Stratagene cyclers from Agilent.
Primer/probe mix with internal control
Each mericon PCR Assay includes rigorously designed primers and probes in a carefully balanced mix that amplify a target sequence and an internal control (IC) with high specificity. This internal control provides information regarding the presence of inhibitors in tested samples and the overall success of the PCR. MAX NHS Ester is employed as the reporter dye for the internal control. With excitation/emission maxima of 524/557 nm and a non-fluorescent quencher (Iowa Black), the MAX dye has a spectral profile similar to HEX, JOE, or VIC, and can be used with most real-time cyclers.