For sensitive and reliable detection of viral RNA/DNA and bacterial DNA, including internal control
Simultaneous detection of pathogen target and internal control
5x master mix for higher sensitivity with more sample input
Correct interpretation of negative results for increased process safety
Clear detection of weak positive target signals
Fast, universal protocol on both standard and fast cyclers
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.
For approximately 200 preps (depending on elution volume): Lyophilized Internal Control RNA, Nucleic Acid Dilution Buffer
Correct interpretation of negative results.
Duplicates of two concentrations of a viral RNA target were co-amplified with the Internal Control in the presence of different amounts of a PCR inhibitory substance (humic acid) on the Rotor-Gene Q. [A] No template controls (NTCs) serve as a reference for Internal Control signal. [B] Amplification of viral RNA target and Internal Control confirms successful amplification. [C] The Internal Control indicates the presence of a low amount of inhibitors. [D] Failure to detect the Internal Control shows the failure of the amplification reaction through presence of inhibitors.
Sensitive detection of BHV-1 on the Rotor-Gene Q.
Bovine herpes virus type 1 was serially diluted (100 to 10-4) and detected by either singleplex real-time PCR or by duplex real-time PCR in parallel with the Internal Control. Real-time PCR was carried out using the QuantiFast Pathogen PCR +IC Kit on the Rotor-Gene Q according to the supplied protocols and without any PCR optimization. The duplex reactions contained a fixed amount of Internal Control template. Each dilution was analyzed in triplicate; one replicate is shown for each dilution.
Sensitive detection of Norovirus.
A Norovirus RNA transcript was serially diluted (100 to 10-5) and detected by either singleplex real-time RT-PCR or by duplex real-time PCR in parallel with the Internal Control. Real-time PCR was carried out using the QuantiFast Pathogen RT-PCR +IC Kit on the Rotor-Gene Q without any PCR optimization. Duplex reactions contained a fixed amount of Internal Control template. Each dilution was analyzed in triplicate; one replicate per dilution is shown.
Sensitive detection of BHV-1 on the ABI 7500.
Bovine herpes virus type 1 was serially diluted (100 to 10-4) and detected by either singleplex real-time PCR or by duplex real-time PCR in parallel with the Internal Control. Real-time PCR was carried out using the QuantiFast Pathogen PCR +IC Kit on the ABI 7500 according to the supplied protocols and without any PCR optimization. The duplex reactions contained a fixed amount of Internal Control template. Each dilution was analyzed in triplicate; one replicate is shown for each dilution.
QIAGEN Internal Control.
Simultaneous extraction and/or amplification of a pathogen target plus an internal positive control will rule out PCR inhibition or other problems that could give a false-negative result, leading to higher process safety. The QIAGEN Internal Control can be added prior to PCR amplification to provide an amplification control, or highly concentrated QIAGEN Internal Control can be added during nucleic acid extraction to provide both an extraction and an amplification control.
High linearity and precision of singleplex and duplex detection.
A 6-log range of both Norovirus RNA singleplex detection and Norovirus/IC duplex detection shows high precision and linearity. Error bars each represent ±1 SD of 3 real-time RT-PCR replicates.
Reliable dilution and storage of RNA standards.
Serial tenfold dilutions of RNA standards (in vitro transcribed RNA) were prepared using either QuantiTect Nucleic Acid Dilution Buffer or RNase-free water, as indicated. These dilutions were used as template in one-step RT-PCR either directly (0 test) or after storage for 2 or 4 weeks at -20°C. Using QuantiTect Nucleic Acid Dilution Buffer resulted in lower CT values and improved stability of the standards.
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 CT values. The buffer ensures longer storage of standards without degradation (see figure "Reliable dilution and storage of RNA standards").
Principle
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 NH4+ 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 NH4+ 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
Procedure
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").
Applications
QuantiFast Pathogen +IC Kits provide sensitive real-time PCR or one-step RT-PCR using sequence-specific probes for detection of pathogen DNA and/or RNA including an internal control. The kits can be used on a wide range of real-time cyclers, including cyclers from QIAGEN, Applied Biosystems, Bio-Rad, Roche (except for capillary cyclers), and Agilent.
Features
Specifications
Applications
Pathogen Detection: Real-time PCR of viral, bacterial or fungal DNA (QuantiFast Pathogen PCR +IC Kit) or one-step RT-PCR for detection of viral RNA (QuantiFast Pathogen RT-PCR +IC Kit)
Reaction type
Real-time PCR or one-step RT-PCR including of an internal control (IC)
For most standard and fast real-time cylcers compatible with duplex PCR/RT-PCR, e.g. Rotor-Gene Q or cyclers from Agilent, Applied Biosystems, BioRad, Roche
With or without ROX
Master Mix is provided without ROX dye, but 2 separate ROX solutions are included: High-ROX Dye Solution for use with ABI cyclers except ABI 7500, ROX Dye Solution (low ROX conc.) for use with ABI 7500 and other suppliers
