Description
The QuantiFast Pathogen +IC Kits are designed for sensitive and rapid real-time PCR or one-step RT-PCR detection of pathogen nucleic acids using sequence-specific probes. To enable high process safety through correct interpretation of negative detection results, each kit contains reagents for multiplex real-time detection of up to 4 user-defined pathogen targets (e.g., virus, bacteria, fungi etc.) plus the Internal Control (IC). Two kit formats are available: The QuantiFast Pathogen RT-PCR +IC Kit for detection of viral RNA, which contains an internal RNA control template, plus the Internal Control primer/probe set, or the QuantiFast Pathogen PCR +IC Kit for detection of viral, bacterial, or fungal DNA, which contains an internal DNA control template, plus the Internal Control primer/probe set. With both kits, ROX is supplied in 2 tubes of different concentrations, enabling use on virtually any real-time instrument. For convenience, the master mix can be stored at 2–8°C.
QuantiFast Pathogen +IC Kits enable simultaneous detection of viral RNA or DNA targets plus the supplied Internal Control over a wide linear range without loss of sensitivity when multiplexing (see figures Sensitive detection of Norovirus on the Rotor-Gene Q and High linearity and precision of singleplex and duplex detection). The protocol has been developed to run fast cycling experiments on most cyclers with high reliability (see figures "Sensitive detection of BHV-1 on the Rotor-Gene Q "and "Sensitive detection of BHV-1 on the ABI 7500"). Combined amplification of pathogen target and the Internal Control using the QuantiFast Pathogen +IC Kits increases process safety of the pathogen detection workflow by ensuring the correct interpretation of negative results (see figure "Correct interpretation of negative results"). QuantiTect Nucleic Acid Dilution Buffer, supplied with the kits, stabilizes RNA and DNA standards during dilution and reaction setup and prevents loss of nucleic acids on plastic surfaces, such as tubes or pipet tips. It enables reliable dilution of standards used to quantify viral nucleic acids, giving a wide linear range, from low to high C T values. The buffer ensures longer storage of standards without degradation (see figure "Reliable dilution and storage of RNA standards").
To enable high process safety through the correct interpretation of negative results, each QuantiFast Pathogen _IC Kit contains reagents for multiplex, real-time detection of a user-defined pathogen target with the Internal Control. Amplifying control and target genes in the same reaction, instead of separate reactions, increases the reliability of gene quantification by minimizing handling errors. QuantiFast Pathogen +IC Kits provide sensitive and rapid real-time multiplex PCR or one-step RT-PCR detection of pathogen nucleic acids on the first attempt (see flowchart "QIAGEN multiplex kits"). The optimized master mix ensures that PCR products in a multiplex reaction are amplified with the same efficiency and sensitivity as PCR products in a corresponding single-amplification reaction. The specially developed fast PCR buffer contains the novel additive Q-Bond, which significantly reduces denaturation, annealing, and extension times (see figure "Fast primer annealing"). A balanced combination of K + and NH 4 + ions as well as unique synthetic Factor MP, promote stable and efficient annealing of primers and probes to the nucleic acid template, enabling high PCR efficiency (see figure "Unique PCR buffer"). In addition, the unique formulation of Sensiscript Reverse Transcriptase ensures highly sensitive reverse transcription of viral RNA, while HotStarTaq Plus DNA Polymerase provides a stringent hot start, preventing the formation of nonspecific products. Components of the QuantiFast Pathogen +IC Kit Kit component Features Benefits 5x QuantiFast Pathogen PCR Master Mix Concentrated master mix Highly concentrated and optimized for sensitive pathogen detection Larger volumes of template can be added to the assay for increased sensitivity HotStarTaq Plus DNA Polymerase 5 min activation at 95ºC Set up of qPCR reactions at room temperature QuantiFast Pathogen Buffer Balanced combination of NH 4 + and K + ions Specific primer annealing ensures reliable PCR results Synthetic Factor MP Reliable multiplexing analysis of up to 4 genes in the same tube Unique Q-Bond additive Faster PCR run times, enabling faster results and more reactions per day Internal Control Assay Internal Control template Internal Control DNA template in the QuantiFast Pathogen PCR +IC Kit A universal DNA amplification control that can be used with different pathogen assays Internal Control RNA in the QuantiFast Pathogen RT-PCR +IC Kit A universal RNA amplification control that can be used with different pathogen assays Internal Control Assay Premixed primer/probe set (TaqMan® probe) labeled with MAX (equivalent to HEX, VIC, etc.) Will not interfere with primers against the pathogen-target Additional kit components QuantiFast Pathogen RT Mix* Contains a unique formulation of Sensiscript Reverse Transcriptase Optimized for highly sensitive detection of pathogen RNA ROX Dye Solution Separate tube of passive reference dye for normalization of fluorescent signals on Applied Biosystems 7500 real-time PCR systems. Optional: Can be used on Stratagene instruments from Agilent Precise quantification on cyclers that require ROX dye. Does not interfere with PCR on any real-time cycler High-ROX Dye Solution Separate tube of passive reference dye for normalization of fluorescent signals on Applied Biosystems 7900 and StepOne real-time PCR systems QuantiTect Nucleic Acid Dilution Buffer Proprietary buffer formulation for dilution and storage of nucleic acid standards Stabilizes RNA and DNA standards during dilution and reaction setup and prevents loss of nucleic acids on plastic surfaces, such as tubes or pipet tips * Included in QuantiFast Pathogen RT-PCR +IC Kits only
QuantiFast Pathogen +IC Kits provide a simple procedure for the detection of a user-defined pathogen and the Internal Control. They contain a ready-to-use master mix for the real-time detection of viral RNA (1-step RT-PCR) or viral, bacterial, and fungal DNA (PCR). There is no need for optimization of reaction and cycling conditions. Simply mix the supplied master mix with the pathogen assay (primers and probe) and the supplied Internal Control Assay and the Internal Control DNA or RNA. Alternatively, if the Internal Control has been added to the sample purification procedure, add RNase-free water instead of Internal Control DNA or RNA to the reaction mix. Then add your sample DNA or RNA and start the reaction on any cycler. The kit handbook contains optimized protocols for use with TaqMan® probes on a wide range of cyclers. It also contains recommendations for selection of dye combinations. Each QuantiFast Pathogen +IC Kit includes the Internal Control Assay and Internal Control DNA or RNA for use as an amplification control via direct addition to the reaction mix. Alternatively, the IC can be added to the purification procedure to control both the purification process and amplification. For addition of the Internal Control to the purification procedure, highly concentrated Internal Control DNA or RNA (High conc.) can be ordered separately (see figure "QIAGEN Internal Control").
