QuantiFast Pathogen +IC Kits

Para la detección sensible y fiable de ARN/ADN vírico y ADN bacteriano, incluido el control interno

S_2537_GEF_QFPatho

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QuantiFast Pathogen RT-PCR +IC Kit (400)

Cat. No. / ID:  211454

Para 400 reacciones de 25 µl: Master Mix, RT Mix, Internal Control Assay liofilizado, Internal Control RNA liofilizado, ROX Dye Solution, High-ROX Dye Solution, RNase-Free Water, Nucleic Acid Dilution Buffer, Buffer TE
1526,00 US$
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KitControl
QuantiFast Pathogen Kit
Internal Control
Type
RT-PCR
PCR
QuantiFast Pathogen +IC Kits están concebidos para su uso en aplicaciones de biología molecular. Estos productos no están concebidos para el diagnóstico, la prevención ni el tratamiento de enfermedades.

✓ 24/7 automatic processing of online orders

✓ Knowledgeable and professional Product & Technical Support

✓ Fast and reliable (re)-ordering

Features

  • Detección simultánea de la diana del microorganismo patógeno y del control interno
  • Mezcla maestra quíntuple para mayor sensibilidad con más entrada de muestra
  • Interpretación correcta de los resultados negativos para una mayor seguridad de los procesos
  • Detección clara de señales de diana positivas débiles
  • Protocolo universal rápido tanto en termocicladores estándar como rápidos

Product Details

Los QuantiFast Pathogen +IC Kits se han diseñado para la detección sensible y rápida, por real-time PCR o por RT-PCR de un paso, de ácidos nucleicos de microorganismos patógenos mediante sondas específicas de secuencia. Para alcanzar un alto nivel de seguridad del proceso a través de la interpretación correcta de los resultados negativos, cada kit contiene reactivos para la detección múltiple en tiempo real de hasta 4 dianas de microorganismos patógenos (p. ej., virus, bacterias, hongos, etc.) más el control interno (IC). Hay dos formatos de kit disponibles: El QuantiFast Pathogen RT-PCR +IC Kit para la detección de ARN vírico, que contiene un molde de control interno de ARN, más el juego de cebadores y sondas de control interno o el QuantiFast Pathogen PCR +IC Kit para la detección de ADN vírico, bacteriano o fúngico, que contiene un molde de control de ADN, más el juego de cebadores y sondas de control interno. En ambos kits, el ROX se suministra en 2 tubos de diferentes concentraciones, lo que permite el uso en cualquier instrumento en tiempo real. Por motivos de conveniencia, la mezcla maestra puede almacenarse a una temperatura de 2-8 °C.

Performance

Los QuantiFast Pathogen +IC Kits permiten la detección simultánea de las dianas de ADN o ARN vírico más el control interno suministrado en un amplio rango lineal sin pérdida de sensibilidad durante el multiplexado (consulte las figuras  Detección sensible de Norovirus en el Rotor-Gene Q y  Gran linealidad y precisión de la detección simple y doble). El protocolo se ha desarrollado para llevar a cabo experimentos de termociclado rápido con gran fiabilidad (consulte las figuras “ Detección sensible del VHB-1 en el Rotor-Gene Q” y “ Detección sensible del VHB-1 en el ABI 7500”). La amplificación combinada de la dianas del microorganismo patógeno y el control interno utilizando los QuantiFast Pathogen +IC Kits aumenta la seguridad del proceso al garantizar la interpretación correcta de los resultados negativos (consulte la figura “ Interpretación correcta de los resultados negativos”).

El QuantiTect Nucleic Acid Dilution Buffer, suministrado con los kits, estabiliza los patrones de ARN y ADN durante la preparación de la reacción y la dilución y previene la pérdida de ácidos nucleicos en las superficies de plástico, como tubos o puntas de pipetas. Permite la dilución fiable de patrones utilizados para cuantificar ácidos nucleicos víricos, lo que ofrece un rango lineal más amplio, desde valores de CT bajos a altos. El patrón garantiza un almacenamiento más prolongado de los patrones sin degradación (consulte la figura “ Almacenamiento y dilución fiables de estándares de ARN”).

See figures

Principle

Para permitir un alto nivel de seguridad del proceso a través de la interpretación correcta de los resultados negativos, cada QuantiFast Pathogen_IC Kit contiene reactivos para la detección múltiple en tiempo real de las dianas de microorganismos patógenos definidas por el usuario con el control interno. La amplificación de genes diana y de control en la misma reacción, en lugar de en reacciones independientes, aumenta la fiabilidad de la cuantificación genética al reducir al mínimo los errores de manipulación.

Los QuantiFast Pathogen +IC Kits ofrecen una detección sensible y rápida, por PCR múltiple en tiempo real o por RT-PCR de un paso, de ácidos nucleicos de microorganismos patógenos en el primer intento (consulte el diagrama de flujo “ Kits de análisis múltiples de QIAGEN”). La mezcla maestra optimizada garantiza que los productos de PCR de una reacción múltiple se amplifiquen con la misma eficacia y sensibilidad que los productos de PCR en la reacción de amplificación simple correspondiente. El tampón de PCR rápida especialmente desarrollado contiene el novedoso aditivo Q-Bond, que reduce de manera significativa los tiempos de desnaturalización, hibridación y extensión (consulte la figura “ Hibridación rápida de cebadores”). Una combinación equilibrada de iones de K+ y NH4+, así como el exclusivo Factor MP sintético, favorecen la hibridación estable y eficaz de cebadores y sondas al molde de ácido nucleico, lo que resulta en una alta eficacia de la PCR (consulte la figura “ Tampón de PCR exclusivo”). Además, la exclusiva formulación de la Sensiscript Reverse Transcriptase garantiza la transcripción inversa muy sensible del ARN vírico, mientras que la HotStarTaq Plus DNA Polymerase proporciona un riguroso hot start, lo que impide la formación de productos no específicos.

Componentes del QuantiFast Pathogen +IC Kit
Componente del kitCaracterísticasBeneficios
5x QuantiFast Pathogen PCR Master MixMezcla maestra concentradaMuy concentrada y optimizada para la detección sensible de microorganismos patógenosSe pueden añadir volúmenes mayores de molde al ensayo para aumentar la sensibilidad
HotStarTaq Plus DNA PolymeraseActivación de 5 min a 95 ºCPreparación de reacciones de qPCR a temperatura ambiente
QuantiFast Pathogen BufferCombinación equilibrada de iones de NH4+ y K+La hibridación de cebadores específicos garantiza la fiabilidad de los resultados de la PCR
Factor MP sintéticoAnálisis múltiple fiable de hasta 4 genes en el mismo tubo
Aditivo exclusivo Q-BondTiempos de análisis por PCR más cortos, lo que produce resultados más rápidos y más reacciones por día
Internal Control AssayMolde de control internoMolde de ADN de control interno del QuantiFast Pathogen PCR +IC KitUn control de amplificación de ADN universal que puede utilizarse con diferentes ensayos de microorganismos patógenos
Internal Control RNA en el QuantiFast Pathogen RT-PCR +IC KitUn control de amplificación de ARN universal que puede utilizarse con diferentes ensayos de microorganismos patógenos
Internal Control AssayJuego de cebadores y sondas premezclado (sonda TaqMan®) etiquetado como MAX (equivalente a HEX, VIC, etc.)No interferirá en los cebadores contra las dianas de microorganismos patógenos
Componentes adicionales del kitQuantiFast Pathogen RT Mix*Contiene una exclusiva formulación de Sensiscript Reverse TranscriptaseOptimizada para la detección muy sensible de ARN de microorganismos patógenos
Solución de colorante ROXTubo separado de colorante de referencia pasivo para la normalización de las señales fluorescentes en sistemas de real-time PCR Applied Biosystems 7500. Opcional: Se puede utilizar en instrumentos Stratagene de AgilentCuantificación precisa en termocicladores que requieren colorante ROX. No interfiere en la PCR en cualquier termociclador en tiempo real
Solución de colorante ROX de alta concentraciónTubo separado de colorante de referencia pasiva para la normalización de las señales fluorescentes en sistemas de real-time PCR Applied Biosystems 7900 y StepOne.
QuantiTect Nucleic Acid Dilution BufferFormulación de tampón patentada para la dilución y el almacenamiento de patrones de ácido nucleicoEstabiliza los patrones de ADN y ARN durante la preparación de la reacción y dilución y previene la pérdida de ácidos nucleicos en las superficies de plástico, como tubos o puntas de pipetas
See figures

Procedure

Los QuantiFast Pathogen +IC Kits proporcionan un procedimiento sencillo para la detección de un microorganismo patógeno definido por el usuario y el control interno. Contienen una mezcla maestra lista para usar para la detección en tiempo real de ARN vírico (RT-PCR de un paso) o de ADN fúngico, bacteriano y vírico (PCR). No es necesario optimizar las condiciones de reacción y termociclado. Simplemente, remueva la mezcla maestra suministrada con el ensayo de microorganismos patógenos (cebadores y sonda), el Internal Control Assay y el Internal Control DNA o RNA. Como alternativa, si el control interno se ha añadido al procedimiento de purificación de muestras, añada agua libre de ARNasas en lugar de Internal Control DNA o RNA a la mezcla de reacción. A continuación, añada su ARN o ADN de la muestra e inicie la reacción en cualquier termociclador. El manual de uso del kit contiene protocolos optimizados para el uso con sondas TaqMan® en una gran variedad de termocicladores. También contiene recomendaciones para la selección de combinaciones de colorantes.

Cada QuantiFast Pathogen +IC Kit contiene el Internal Control Assay y el Internal Control DNA o RNA para su uso como un control de amplificación mediante adición directa a la mezcla de la reacción. En su defecto, el IC puede añadirse al procedimiento de purificación para controlar tanto el proceso de purificación como la amplificación. Para añadir el control interno al procedimiento de purificación, se pueden pedir por separado Internal Control DNA o RNA (alta conc.) (consulte la figura “ Control interno de QIAGEN”).

See figures

Applications

Los QuantiFast Pathogen +IC Kits proporcionan RT-PCR de un paso o real-time PCR sensibles mediante el uso de sondas específicas de secuencia para la detección simultánea de ADN y/o ARN de microorganismos patógenos, incluido un control interno. Los kits pueden utilizarse en una gran variedad de termocicladores en tiempo real, incluidos los termocicladores de QIAGEN, Applied Biosystems, Bio-Rad, Roche (excepto los termocicladores de capilares) y Agilent.

Supporting data and figures

Specifications

FeaturesSpecifications
ApplicationsDetección de microorganismos patógenos: Real-time PCR para ADN vírico, bacteriano o fúngico (QuantiFast Pathogen PCR +IC Kit) o RT-PCR de un paso para la detección de ARN vírico (QuantiFast Pathogen RT-PCR +IC Kit)
Sample/target typeQuantiFast Pathogen PCR +IC Kit: ADN vírico, bacteriano o fúngico; QuantiFast Pathogen RT-PCR +IC Kit: ARN vírico
Single or multiplexDoble
Reaction typeReal-time PCR o RT-PCR de un paso que incluye un control interno (IC)
Real-time or endpointTiempo real
SYBR Green I or sequence-specific probesSondas específicas de secuencia
Thermal cyclerPara la mayoría de los termocicladores en tiempo real rápidos y estándar con PCR/RT-PCR doble; p. ej., Rotor-Gene Q o termocicladores de Agilent, Applied Biosystems, BioRad, Roche
With or without ROXLa mezcla maestra se suministra sin contraste ROX, pero se incluyen 2 soluciones ROX: Solución de colorante ROX en alta concentración para usar con termocicladores ABI 7500, solución de colorante ROX (baja concentración de ROX) para usar con ABI 7500 y otros proveedores

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
How do I setup and validate a multiplex PCR assay with QIAGEN PCR kits?

Ensure PCR amplicons are as short as possible, ideally 60–150 bp. Always use the same algorithm or software to design the primers and probes. For optimal results, only combine assays that have been designed using the same parameters.

 

Check the functionality of each set of primers and probes in individual assays before combining the different sets in the multiplex assay. Choose compatible reporters and quenchers based on a specific instrument. See How do I select appropriate reporter and quencher combinations for multiplex PCR.

 

FAQ ID -9093
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
Can I use uracil-N-glycosylase (UNG) with the QuantiFast and Rotor-Gene PCR kits?

No. UNG treatment does not provide any advantage for the QuantiFast and Rotor-Gene PCR kits because the mastermixes do not contain dUTP. Use the QuantiTect kits if you intend to use the UNG treatment.

FAQ ID -9092
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
Why do I see multiple high-intensity peaks in my qPCR dissociation curve at temperatures less than 70ºC?

If the extra peaks seem irregular or noisy, do not occur in all samples, and occur at temperatures less than 70 ºC, then these peaks may not represent real PCR products and instead may represent artifacts caused by instrument settings.

 

Usually extra peaks caused by secondary products are smooth and regular, occur reproducibly in most samples, and occur at temperatures greater than 70 ºC. Characterization of the product by agarose gel electrophoresis is the best way to distinguish between these cases. If only one band appears by agarose gel then the extra peaks in the dissociation curve are instrument artifacts and not real products. If this is the case, refer to the thermal cycler user manual, and confirm that all instrument settings (smooth factor, etc.) are set to their optimal values.

 

FAQ ID -90990
How do I quantify gene expression levels if the amplification efficiencies are different between the genes of interest and endogenous reference gene?

The REST 2009 (Relative Expression Software Tool) software applies mathematic models that compensate for the different PCR efficiencies of the gene of interest and reference genes. In addition, the software can use multiple reference gene normalization to improve the reliability of result, as well as provides statistical information suitable for robust comparison of expression in groups of treated and untreated. QIAGEN offers the REST 2009 software free of charge.

FAQ ID -9095
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
What do I do if no fluorescent signal is detected in a real-time PCR assay?

Check the template quality and integrity by amplifying an endogenous control gene. Check the amplicon by QIAxcel Advanced system or agarose gel electrophoresis to show that amplification was successful.

 

Determine whether the gene of interest is expressed in your sample. See How can I find out if my gene of interest is express in a specific tissue type or cell line.  Ensure the assay setup and cycling conditions are correct, and that the data collection channel matches the emission wavelength of the fluorescent dye used. Use a control sample in which the gene of interest is definitely expressed.

 

If the issue persists, please send the original run file to QIAGEN Technical Services.

FAQ ID -9091
How do I select appropriate reporter and quencher combinations for multiplex PCR?

For duplex analysis, using non-fluorescent quenchers (e.g., Black Hole Quencher®) is preferred over fluorescent quenchers (e.g., TAMRA fluorescent dye). For triplex and 4-plex analysis, QIAGEN strongly recommends using non-fluorescent quenchers. Generally, use the green channel, the yellow channel, and the orange and crimson channels to detect the least abundant target, the second least abundant target, and the two most abundant targets, respectively. For instrument-specific recommendations, please see the handbooks for the QuantiTect Multiplex PCR kit, QuantiFast Multiplex kit or Rotor-Gene Multiplex kit.

 

FAQ ID -9094
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
Can I skip the gDNA wipeout buffer treatment step for the QuantiTect Reverse Transcription Kit?

The gDNA wipeout buffer incubation step can be skipped when the total RNA is free from genomic DNA. However, the gDNA wipeout buffer is still required to be added because the reverse transcription step is optimized in the presence of components in the gDNA wipeout buffer.

FAQ ID -9098
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
How should I handle and store absolute quantitation standards for real-time experiments?
Store the standards at a high concentration in aliquots at -20oC to -70oC. If using low concentrations, stabilize standards with carrier nucleic acid. It is always best to use freshly diluted standards for each experiment. If possible, use siliconized tubes for standard (and target) dilutions. This will prevent any unspecific binding of nucleic acids to the plastic.
FAQ ID -9099
How do I ensure reliable results for High Resolution Melting (HRM) assays?

Reliable HRM analysis results depend on template quality, highly specific HRM PCR kit with a saturation dye, a real-time instrument with HRM capability, and powerful software package. Factors critical for successful HRM analysis are:

 

  • Use the same genomic DNA purification procedure for all samples being analyzed by HRM. This avoids variation due to differing composition of elution buffers.
  • DNA template concentrations should be normalized using the same dilution buffer. Ensure the CT values are below 30 and differ no more than 3 CT values across individual samples.
  • Design assays with amplicon length 70–350 bp. For SNP analysis, use amplicon length 70–150 bp.
  • Always start with 0.7 µM primer concentration

 

For more details, please refer to the HRM Technology – FAQs and the Critical Success Factor for HRM performance.

FAQ ID -9097
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 do I avoid collecting a fluorescence reading from primer-dimer with the QuantiTect SYBR Green PCR Kit?

Depending on primer design and copy number of target, primer-dimer may occur and its signal might be detected. Typical strategies against this are to optimize PCR conditions and/or redesign the assay.

 

Alternatively, an additional data-acquisition step can be added to the 3-step cycling protocol. First, determine the melting temperatures (Tm) for both the amplicon and the primer-dimer. Then, add a 15 second data-acquisition step with a temperature that is higher than the primer-dimer Tm, but approximately 3ºC lower than the specific amplicon Tm.

FAQ ID -9096
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