QuantiFast Pathogen +IC Kits

For sensitive and reliable detection of viral RNA/DNA and bacterial DNA, including internal control

Features

  • 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
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QuantiFast Pathogen RT-PCR +IC Kit (400)

Cat. No. / ID: 211454

For 400 x 25 µl reactions: Master Mix, RT Mix, lyophilized Internal Control Assay, lyophilized Internal Control RNA, ROX Dye Solution, High-ROX Dye Solution, RNase-Free Water, Nucleic Acid Dilution Buffer, Buffer TE
KitControl
QuantiFast Pathogen Kit
Internal Control
Type
RT-PCR
PCR
QuantiFast Pathogen +IC Kits are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.
✓ 24/7 automatic processing of online orders ✓ Knowledgeable and professional Product & Technical Support ✓ Fast and reliable (re)-ordering

Product Details

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.

Performance

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 CT values. The buffer ensures longer storage of standards without degradation (see figure " Reliable dilution and storage of RNA standards").

See figures

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
See figures

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").

 

See figures

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.

Supporting data and figures

Specifications

FeaturesSpecifications
ApplicationsPathogen 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)
Sample/target typeQuantiFast Pathogen PCR +IC Kit: viral, bacterial or fungal DNA; QuantiFast Pathogen RT-PCR +IC Kit: viral RNA
Single or multiplexDuplex
Reaction typeReal-time PCR or one-step RT-PCR including of an internal control (IC)
Real-time or endpointReal-time
SYBR Green I or sequence-specific probesSequence-specific probes
Thermal cyclerFor 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 ROXMaster 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

FAQ

What is the detection limit for the QuantiFast Pathogen + IC kits?
The detection limit for the QuantiFast Pathogen RT-PCR and PCR kits is < 10 copies.
FAQ ID -2453
What is the nature of the Internal Control in the QuantiFast Pathogen + IC kit?
The DNA IC is a non-linearized plasmid. The RNA IC is an in vitro transcript. Both are naked nucleic acids. The size (base pair length) of both templates is sufficient to allow for efficient purification with standard methods for nucleic acid extraction.
FAQ ID -2450
Why do replicates in real-time PCR have different plateau heights?

Replicates in real-time PCR may have different plateau heights due to differences in the reaction kinetics for each sample. Even though replicates start out with identical template amounts, the rate at which reagents are being depleted, and the point when exponential accumulation of PCR product stops and becomes linear, differ between replicates. This will result in different plateau heights, the stage where PCR reactions have come to a halt, and little or no additional PCR product is being amplified. You can find further information in Chapter 'Quantification of target amounts' of our Brochure "Critical Factors for Successful Real-Time PCR".

 

FAQ ID -539
Do the QuantiFast Pathogen PCR +IC Kit and the QuantiFast Pathogen RT-PCR +IC Kit contain ROX in the master mix?
The QuantiFast Pathogen PCR +IC Kit and the QuantiFast Pathogen RT-PCR +IC Kit are supplied with master mixes that do not contain ROX. Instead, 2 different ROX concentrations are supplied in separate tubes. “High-ROX Dye Solution” is suitable for use with all ABI cyclers except ABI 7500, and “ROX Dye Solution” is suitable for use with ABI 7500 and, optionally, instruments from Stratagene (Agilent). Recommendations are provided in the handbook.
FAQ ID -2605
How long does a QuantiFast Pathogen + IC run take on the Rotor-Gene Q?

Using the QuantiFast Pathogen + IC kits on the Rotor-Gene Q:

 

RT-PCR

 
40 cycles ~95 minutes
45 cycles ~100 minutes

 

 

PCR  
40 cycles ~75 minutes
45 cycles ~80 minutes

FAQ ID -2452
On which cyclers can the QuantiFast Pathogen PCR +IC Kit or the QuantiFast Pathogen RT-PCR +IC Kit be used?
The QuantiFast Pathogen PCR + RT-PCR +IC Kits can be used on all leading block cycler platforms, but not on capillary systems (e.g., LightCycler 2.0).
FAQ ID -2596
What should the cycler set-up be for a duplex reaction with the pathogen assay (FAM) and the Internal Control assay (MAX) on ABI instruments, when using the QuantiFast Pathogen PCR +IC Kit or the QuantiFast Pathogen RT-PCR +IC Kit?
No calibration for MAX is needed if the instrument is calibrated for VIC. Use the FAM/SYBR Green filter for the pathogen assay and the VIC/JOE filter for the IC assay. To detect the Internal Control (MAX) in the VIC/JOE filter, create a new detector (e.g., “MAX/IowaBlack”). Assign “VIC” as the reporter dye and “None” for the quencher dye.
FAQ ID -2610
What should the Rotor-Gene Q cycler settings be for a duplex reaction with the pathogen assay (FAM) and the Internal Control assay (MAX), when using the QuantiFast Pathogen PCR +IC Kit or the QuantiFast Pathogen RT-PCR +IC Kit?
Please follow the instructions in the handbook. Briefly, use gain optimization “Before First Acquisition” for the pathogen assay (FAM) in the Green channel, and use a fixed gain of 9 for the Internal Control Assay (MAX) in the Yellow channel.
FAQ ID -2608
When performing the Internal Control assay for the QuantiFast Pathogen PCR +IC Kit and the QuantiFast Pathogen RT-PCR +IC Kit, what channel or filter can be used to detect MAX?
MAX can be detected using the same channels or filters as for HEX, JOE, or VIC.
FAQ ID -2597
What is the threshold cycle or Ct value?
The Ct or threshold cycle value is the cycle number at which the fluorescence generated within a reaction crosses the fluorescence threshold, a fluorescent signal significantly above the background fluorescence. At the threshold cycle, a detectable amount of amplicon product has been generated during the early exponential phase of the reaction. The threshold cycle is inversely proportional to the original relative expression level of the gene of interest.
FAQ ID -2682
Are the Internal Controls used in the QuantiFast Pathogen PCR +IC Kit and the QuantiFast Pathogen RT-PCR +IC Kit homologous to a known target?
No, the Internal Controls used in the QuantiFast Pathogen PCR +IC Kit and the QuantiFast Pathogen RT-PCR +IC Kit are an artificial sequence that is not present in biological sample material.
FAQ ID -2598
Can the QuantiFast Pathogen PCR +IC Kit or the QuantiFast Pathogen RT-PCR +IC Kit be used with hybridization probes?

No, the QuantiFast Pathogen PCR +IC kits are designed for use with hydrolysis probes (also known as TaqMan® probes) only.

See trademarks

FAQ ID -2595
When using the QuantiFast Pathogen PCR +IC Kit or the QuantiFast Pathogen RT-PCR +IC Kit, what should the fluorescent label of the probe be for the customer-defined assay to detect the pathogen?
Typically, the target-specific probe should be labeled with FAM as the reporter dye and a non-fluorescent quencher (e.g., Dark Quencher, Black Hole Quencher [BHQ] or Iowa Black Quencher). Other reporter dyes than FAM, detected in a different detection channel than MAX, may also be suitable. It is not recommended to use fluorescent quenchers (e.g., TAMRA fluorescent dye). Due to their own native fluorescence, fluorescent quenchers contribute to an overall increase in background and reduce the signal-to-noise ratio.
FAQ ID -2594
Can the Internal Control DNA or RNA be added directly to the sample?
No, the Internal Control template DNA or RNA must be added to the lysis buffer or to the lysate in order to prevent the loss of Internal Control template thorough matrix effects.
FAQ ID -2602
What should the cycler set-up be for a duplex reaction with the pathogen assay (FAM) and the Internal Control assay (MAX) on Mx instruments from Stratagene (Agilent), when using the QuantiFast Pathogen PCR +IC Kit or the QuantiFast Pathogen RT-PCR +IC Kit?
In the “Filter Gain Settings” dialog box, set the filter gain to a value of 4 for both the FAM/SYBR Green and HEX/JOE/VIC filters. See the Mx instrument/software manual for details.
FAQ ID -2609
Why does my realtime PCR assay quality decrease over time?
Make sure that template, primers, probes, and amplification reagents are stored correctly and avoid multiple freeze–thaw cycles for oligonucleotides and template. Check the performance of your real-time instrument as some instruments require the halogen lamp to be frequently replaced. Lasers must also be replaced occasionally.
FAQ ID -589
Which DNA/RNA extraction kits were tested in combination with the QuantiFast Pathogen PCR +IC Kit and the QuantiFast Pathogen RT-PCR +IC Kit?
FAQ ID -2606
Does the amount of Internal Control to be spiked into the lysis buffer or lysate depend on the elution volume only?

Principally, yes. However, additional factors influencing the CT are:

1. Extraction efficiency, which depends on extraction method and sample material.

2. Volume of the eluate used for PCR.

Examples: Use of the Internal Control according to handbook instructions (0.1µl per 1µl elution volume) for two different workflows.

  • Workflow 1: High extraction efficiency: use of 5 µl of the eluate for PCR results in a CT value of 29-30.
  • Workflow 2: High extraction efficiency: use of 10 µl of the eluate for PCR results in a CT of 28-29.

Depending on the workflow, the amount of Internal Control to be added to the extraction might have to be adjusted to more than 0.1 µl per 1 µl elution volume, or to less than 0.1 µl per 1 µl elution volume, in order to achieve a CT value within the expected range.

Running the Internal Control DNA/RNA provided with the QuantiFast Pathogen +IC Kit in a separate reaction can serve as a reference for adjusting the amount of Internal Control DNA/RNA (High conc.) to be added to the extraction.

FAQ ID -2604
During data analysis, how should the threshold be set in the Yellow channel on the Rotor-Gene Q cycler to analyze the Internal Control from the QuantiFast Pathogen PCR +IC Kit or the QuantiFast Pathogen RT-PCR +IC Kit
On the Rotor-Gene Q, setting the threshold in the Yellow channel to an absolute value of 0.05 will give satisfactory results in most cases.
FAQ ID -2607
What is the difference between QuantiTect Virus Kit and the new QuantiFast Pathogen +IC Kits?
The QuantiTect Virus Kit and the new QuantiFast Pathogen +IC Kits are based on similar reaction chemistry and show identical sensitivity but as a new feature, the QuantiFast Pathogen kits include an Internal Control template and the corresponding primer/probe set for duplex amplification of a user-defined pathogen target with the provided Internal Control. Additional new features are pack size, kit variants, ROX variants, and speed.
FAQ ID -2448
What are the labels of the probe which is used in the Internal Control Assay for detection of the IC?
In the QuantiFast Pathogen + IC kit, the probe is labeled with MAX-NHS ester (MAX) as the reporter dye which has a spectrum equivalent to HEX, JOE or VIC dyes. IowaBlack is used as the quencher dye. IowaBlack is a non-fluorescent quencher (dark quencher).
FAQ ID -2449
Can the Internal Controls be ordered separately in the QuantiFast Pathogen + IC kit for use during purification?

Yes, the Internal Control RNA (High conc.) and Internal Control DNA (High conc.) templates are available under a separate catalog number. After reconstitution according to the description in the handbook, these IC templates have a 10x higher concentration than the Internal Control templates provided in the kits.   They are sufficiently concentrated to be spiked into the sample prep without replacing too much of the sample input volume.

 

**Please note that there is no assay (primer/probe set) for amplification of the IC included in these separate catalog numbers. The assay is only included in the QuantiFast Pathogen PCR +IC Kit and QuantiFast Pathogen RT-PCR +IC Kit.  The ICs cannot be used with the QuantiTect Virus kits because the assay (primer/probe set) for the detection of the IC is provided with the QuantiFast Pathogen Kits only.

 

FAQ ID -2451
Why do the Internal Control templates for extraction (Internal Control DNA or RNA [High conc.]) have a 10x higher concentration than the IC templates provided with the QuantiFast Pathogen PCR +IC Kit and the QuantiFast Pathogen RT-PCR +IC Kit?

After reconstitution according to handbook instructions, the Internal Control (IC) templates provided with the kits have a ready-to-use concentration for direct use as an amplification control in PCR or RT-PCR. For each 25 µl reaction, 2.5 µl of the IC are added, resulting in an expected CT value of approximately 29-30.

In order to achieve the same CT value when using the IC as an extraction control, more IC template has to be spiked in before extraction. The exact amount depends mainly on the elution volume. For each 1 µl of elution volume, 0.1 µl of the IC High conc. should be added to each of the extraction samples. This should result in a CT value in the PCR or RT-PCR reactions of approximately 29-30.

 

Examples: For 100 µl of elution volume, 10 µl of the IC, per sample, should be added to the lysis buffer or lysate. For 50 µl of elution volume, 5 µl of the IC, per sample, should be added to the lysis buffer or lysate.

FAQ ID -2603
How many times can the Internal Controls used in the QuantiFast Pathogen PCR +IC Kit and the QuantiFast Pathogen RT-PCR +IC Kit be freeze-thawed?
The Internal Control templates can be freeze-thawed for up to 6 times without decrease in performance. However, great care should be taken to avoid inadvertently introducing RNAses/DNAses into the Internal Control template solutions. In general, in order to avoid repeated freeze-thaw cycles, we recommend preparing aliquots of the Internal Control templates.
FAQ ID -2599
What is the difference between adding the Internal Control (IC) template used in the QuantiFast Pathogen PCR +IC Kit and the QuantiFast Pathogen RT-PCR +IC Kit to the amplification reaction versus adding the IC template at the extraction step?
When the IC is added to the amplification reaction mix, the IC signal confirms that PCR amplification has been successful. When it is added at the extraction step to the lysis buffer or lysate, the IC signal confirms that nucleic acid purification and PCR amplification have been successful.
FAQ ID -2600
For the QuantiFast Pathogen RT-PCR +IC kit, does the Internal Control also control for the reverse transcription reaction?
Yes, the Internal Control RNA will only give the expected signal when both the reverse transcription and the PCR reactions have been successful.
FAQ ID -2601