QuantiFast Pathogen +IC Kits

ウイルスRNA/DNAやバクテリアDNAとインターナルコントロールの高感度検出

S_2537_GEF_QFPatho

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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
PLN 8,965.00
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KitControl
QuantiFast Pathogen Kit
Internal Control
Type
RT-PCR
PCR
QuantiFast Pathogen +IC Kitsは分子生物学的アプリケーション用であり、疾病の診断、予防、あるいは治療に使用することはできません。

✓ オンライン注文による24時間年中無休の自動処理システム

✓ 知識豊富で専門的な製品&テクニカルサポート

✓ 迅速で信頼性の高い(再)注文

特徴

  • 病原体ターゲットとインターナルコントロールを同時に検出
  • 5x マスターミックスでサンプル添加量を増加でき感度を増大
  • 陰性結果の明確化により、病原体の新規アッセイ系を簡便に構築
  • 低い陽性シグナルも明確に検出
  • スタンダードおよび高速サイクラーに対応する迅速な共通プロトコール

製品詳細

QuantiFast Pathogen +IC Kits は、配列特異的なプローブを用いたリアルタイムPCR あるいは1 ステップRT-PCR により、病原体核酸を高感度かつ高速に検出するために特化されています。偽陰性を排除した確実性の高い系を実現するために、最高4種類の研究対象の病原体ターゲット(ウイルス、バクテリア、真菌など)とインターナルコントロール(IC)をリアルタイムに検出するための試薬が各キットに入っています。キットフォーマットは2種類あります:Internal RNA Controlテンプレートとこれを検出するInternal Controlプライマー/プローブセットが入ったウイルスRNA 検出用QuantiFast Pathogen RT-PCR +IC Kit、あるいはInternal DNA Controlテンプレートとこれを検出するInternal Control プライマー/プローブセットが入ったウイルス/バクテリア/真菌DNA 検出用 QuantiFast Pathogen PCR +IC Kit。両キットともに2種類の濃度のROX(チューブ)がキットに入っているので、どのようなリアルタイムサイクラーにも使用可能です。マスターミックスは2~8 ℃で保存でき、簡便に取り扱えます。

パフォーマンス

QuantiFast Pathogen +IC Kitsを用いたマルチプレックスアッセイは、感度を損なうことなく複数のウイルスRNAやDNA ターゲットと添付のインターナルコントロールを同時に幅広い範囲で正確に検出できます(図” Rotor-Gene Qでノロウイルスの高感度検出”および“ Singleplexと Duplex検出で高い直線性と精度を実現”)。本プロトコールはほとんどのサイクラーで迅速なサイクリング実験を高い信頼性で行なうために開発されました(図“ Rotor-Gene QでBHV-1の高感度検出”および“ ABI 7500でBHV-1の高感度検出”)。QuantiFast Pathogen +IC Kitsを用いて病原体ターゲットとインターナルコントロールの増幅を組み合わせることにより、陰性結果を明確に判定できるので、確実な病原体検出用のワークフローを実現します(図“ 陰性結果の正確な判定”)。

キットに同梱のQuantiTect Nucleic Acid Dilution Bufferは、希釈および反応セットアップ中にRNAやDNAスタンダードを安定化し、チューブやピペットチップの様なプラスチック表面への核酸の吸着を防ぎます。このバッファーによりウイルス核酸の定量に用いるスタンダードを正確に希釈でき、広範囲のCT 値において高い直線性が得られます。本バッファーはスタンダードの分解を防ぎ、長期保存を可能にします(図 “ RNA スタンダードの正確な希釈および保存”)。

図参照

原理

陰性結果を明確に判定でき確実性の高い系を実現するため、インターナルコントロールと対象とする病原体の標的遺伝子を同時に検出するマルチプレックスリアルタイム検出用試薬が各QuantiFast Pathogen +IC Kitに入っています。別々の反応ではなく同一反応内でコントロール遺伝子と標的遺伝子を増幅することで、マニュアルでの作業によるエラーが最小限に抑えられ、遺伝子定量の信頼性が高くなります。

QuantiFast Pathogen +IC Kitsは、最初の実験でマルチプレックスリアルタイムPCRや1ステップ RT-PCRによる高感度で迅速な病原体核酸検出を実現します(フローチャート“ QIAGEN Multiplex Kit”)。至適化されたマスターミックスにより、マルチプレックス反応でのPCR産物をシングル増幅反応に相当するPCR産物と同様の効率および感度で確実に増幅します。特別に開発された高速PCR用バッファーは、変性、アニーリングおよびエクステンション時間も顕著に短縮する斬新なQ-Bondを含有しています(図“ プライマーの高速なアニーリング”)。画期的な合成添加剤Factor MPとK+およびNH4+のイオン配合比により、プライマーとプローブが核酸テンプレートに効率的かつ安定してアニーリングし、高いPCR効率と感度を実現します(図“ ユニークなPCRバッファー”)。さらにSensiscript Reverse Transcriptaseのユニークな組成はウイルスRNAの高感度な逆転写を実現し、HotStarTaq Plus DNA Polymeraseは厳密なホットスタートにより非特異的な産物の生成を回避します。

QuantiFast Pathogen +IC Kitの成分
成分 特長 利点
5x QuantiFast Pathogen PCR Master Mix 高濃度マスターミックス 高感度な病原体検出用に高濃度で至適化済み テンプレート量を増やしアッセイ感度の増大を実現
HotStarTaq Plus DNA Polymerase 95℃、5分の活性化 室温で定量PCRのセットアップ
QuantiFast Pathogen Buffer NH4+とK+イオンの配合バランス 特異性の高いプライマーのアニーリングで信頼性の高いPCR結果
合成添加剤Factor MP 同一チューブ中の最高4遺伝子を高い信頼性でマルチプレックス解析
ユニークな添加剤Q-Bond PCR反応時間が短縮されるため迅速に結果が得られ、1日あたりの反応数を増やせる
Internal Control Assay インターナルコントロールテンプレート QuantiFast Pathogen PCR +IC KitのInternal Control DNAテンプレート 異なる病原体アッセイとともに使用できるユニバーサルなDNA増幅コントロール
QuantiFast Pathogen RT-PCR +IC KitのInternal Control RNA 異なる病原体アッセイとともに使用できるユニバーサルなRNA増幅コントロール
Internal Control Assay MAX(HEX、VICなどに相当)で標識したプレミックスのプライマー/プローブセット(TaqManプローブ) 標的病原体に対するプライマーを阻害しない
追加のキット成分 QuantiFast Pathogen RT Mix* ユニークな組成のSensiscript Reverse Transcriptase 病原体RNAの高感度検出用に至適化済み
ROX Dye Solution Applied Biosystems 7500リアルタイムPCRシステムでの蛍光補正用にpassive reference dye が別途チューブ包装。オプション:Agilent、Stratagene 装置で使用可能 ROX色素が必要なサイクラーで正確な定量。どのリアルタイムサイクラーでもPCRを妨害しない
High-ROX Dye Solution Applied Biosystems 7900およびStepOneリアルタイムPCR装置での蛍光補正用にpassive reference dyeが別途チューブ包装
QuantiTect Nucleic Acid Dilution Buffer 核酸スタンダードの希釈および保存に最適な独自のバッファー組成 希釈や反応セットアップ中のRNAやDNAスタンダードを安定化し、チューブやピペットチップのようなプラスチック表面への核酸の吸着を防止
*QuantiFast Pathogen RT-PCR +IC Kit にのみ同梱
図参照

操作手順

QuantiFast Pathogen +IC Kitsは研究対象の病原体とインターナルコントロールの検出を簡単な操作で実現します。本キットは即使用可能なマスターミックスを含み、ウイルスRNA(1ステップRT-PCR)あるいはウイルス/バクテリア/真菌DNA(PCR)のリアルタイム検出を実現します。反応条件やサイクリング条件の至適化は不要です。マスターミックスに病原体アッセイ(プライマーとプローブ)とInternal Control Assay およびInternal Control DNA/RNAをミックスするだけです。あるいは、Internal Control DNA/RNAを核酸精製プロセスで添加した場合は、Internal Control DNA/RNAの代わりにRNase フリー水を反応ミックスに添加します。その後、DNA あるいはRNA テンプレートを添加し、サイクラーで反応を開始します。ハンドブックにはTaqMan プローブ用に至適化されたプロトコールが入っており、様々なサイクラーで使用可能です。また推奨する色素の組み合わせも記載されています。

各QuantiFast Pathogen +IC Kits に入っている Internal Control Assay およびInternal Control DNA/RNA は、反応ミックスに直接添加して増幅コントロールとして使用します。あるいは核酸精製プロセスおよび増幅の両方をコントロールするために、インターナルコントロールを核酸精製プロセスで添加することも可能です。精製プロセスでインターナルコントロールを添加する場合は、高濃度のInternal Control DNA あるいはRNA(High conc.)を別途注文できます(図“ QIAGEN Internal Control”)。

 

図参照

アプリケーション

QuantiFast Pathogen +IC Kitsは配列特異的なプローブを用いた高感度なリアルタイムPCRあるいは1ステップRT-PCRでインターナルコントロールを含む病原菌DNA/RNAの検出を実現します。本キットはQIAGEN、Applied Biosystems、Bio-Rad、Roche(キャピラリー式サイクラーを除く)、Agilent製サイクラーを含む様々なリアルタイム用サーマルサイクラーで使用可能です。

裏付けデータと数値

Specifications

FeaturesSpecifications
ApplicationsJAPathogen 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

リソース

MSDS (2)
Download Safety Data Sheets for QIAGEN product components.
Download Safety Data Sheets for QIAGEN product components.
パンフレット (1)
Now with even more applications!
Certificates of Analysis (1)

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