QuantiFast Pathogen + IC Kits

内部コントロールを含むウイルスRNA/DNAおよび細菌DNAの高感度かつ信頼性の高い検出

S_2537_GEF_QFPatho

✓ 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

QuantiFast Pathogen RT-PCR +IC Kit (400)

Cat. No. / ID:   211454

400 x 25 µl反応の場合: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内部コントロールを同時検出
  • より多くのサンプルインプットでより高い感度が得られる5xマスターミックス
  • プロセス安全性を高める陰性結果の正しい解釈
  • 弱い陽性ターゲットシグナルを明確に検出
  • 標準サイクラーと高速サイクラーの両方で、迅速なユニバーサルプロトコール

製品詳細

QuantiFast Pathogen +IC Kitsは、配列特異的プローブを使用して、病原体核酸を高感度で迅速に、リアルタイムPCRまたは1ステップRT-PCR検出ができるように設計されています。陰性検出結果を正しく解釈することで高いプロセス安全性を可能にするため、各キットには最大4つのユーザー定義の病原体ターゲット(ウイルス、細菌、真菌など)とInternal Control(IC)のマルチプレックスリアルタイム検出を行う試薬が含まれています。キットには2つの形式があります。ウイルスRNA検出用のQuantiFast Pathogen RT-PCR +IC Kit(内部RNAコントロールテンプレートとInternal Controlプライマー/プローブセットを含む)、またはウイルス、細菌、真菌DNA検出用のQuantiFast Pathogen PCR +IC Kit(内部DNAコントロールテンプレートとInternal Controlプライマー/プローブセットを含む)です。どちらのキットでも、ROXは濃度の異なる2本のチューブで提供され、事実上どのリアルタイムPCR装置でも使用できます。マスターミックスは2~8℃で保存できるので便利です。

パフォーマンス

QuantiFast Pathogen +IC Kitsは、マルチプレックス時に感度を損なうことなく、広い直線範囲にわたってウイルスRNAまたはDNAターゲットと付属のInternal Controlを同時に検出できます(図 Rotor-Gene Qでのノロウイルスの高感度検出および シングルプレックスおよびデュプレックス検出の高い直線性と精度参照)。このプロトコールは、ほとんどのサイクラーで、高速サイクリング実験を高い信頼性で実行できるように開発されています(図「 Rotor-Gene QでのBHV-1の高感度検出」および「 ABI 7500でのBHV-1の高感度検出」参照)。QuantiFast Pathogen +IC Kitsを使用して病原体ターゲットとInternal Controlを組み合わせて増幅すると、陰性結果の正しい解釈が保証され、病原体検出ワークフローのプロセス安全性が向上します(図「 陰性結果の正しい解釈」参照)。

キットに付属するQuantiTect Nucleic Acid Dilution Bufferは、希釈および反応セットアップ中にRNAおよび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での核酸の損失を防ぎます。ウイルス核酸定量に使用する標準の信頼性の高い希釈を可能にし、低~高CT値の広い直線範囲が得られます。このバッファーは、変性することなくスタンダードを長期保存できます(図「 信頼性の高いRNA標準の希釈と保存」参照)。

図参照

原理

陰性結果を正しく解釈することで高いプロセス安全性を可能にするため、各QuantiFast Pathogen _IC Kitには、ユーザー定義の病原体ターゲットとInternal Controlをマルチプレックスでリアルタイム検出するための試薬が含まれています。コントロール遺伝子と標的遺伝子を別々の反応ではなく、同じ反応で増幅することで、ハンドリングエラーが最小限に抑えられ、遺伝子定量の信頼性が向上します。

QuantiFast Pathogen +IC Kitsは、病原体核酸の高感度かつ迅速なリアルタイムマルチプレックスPCRまたは1ステップ RT-PCR検出を最初の試みで実現します(フローチャート「 QIAGENマルチプレックスキット」参照)。最適化されたマスターミックスは、マルチプレックス反応時にPCR産物が、対応するシングル増幅反応時のPCR産物と同じ効率と感度で増幅されることを保証します。特別に開発された高速PCRバッファーには、変性、アニーリング、伸長時間を大幅に短縮する新しい添加剤Q-Bondが含まれています(図「 高速プライマーアニーリング」参照)。K+イオンとNH4+イオンのバランスのとれた組み合わせと独自の合成Factor MPにより、プライマーとプローブの核酸テンプレートへの安定的かつ効率的なアニーリングが促進され、高いPCR効率が可能になります(図「 独自のPCRバッファー」参照)。さらに、Sensiscript Reverse Transcriptaseの独自の組成により、ウイルスRNAの高感度逆転写が保証され、HotStarTaq Plus DNA Polymeraseにより厳密なホットスタートが提供され、非特異的産物の形成が防止されます。

QuantiFast Pathogen +IC Kitのコンポーネント
キットのコンポーネント特徴メリット
5x QuantiFast Pathogen PCR Master Mix濃縮マスターミックス病原体を高感度で検出できるように高濃度で最適化感度を上げるため、より多くのテンプレート量をアッセイに添加可能
HotStarTaq Plus DNA Polymerase95ºC 5分で活性化室温でqPCR反応をセットアップ
QuantiFast Pathogen BufferNH4+イオンとK+イオンのバランスの取れた組み合わせ特異的なプライマーアニーリングにより信頼性の高いPCR結果を保証
合成Factor MP同じチューブで最大4つの遺伝子を高い信頼性でマルチプレックス分析
独自のQ-Bond添加剤PCRの実行時間が短縮され、より迅速に結果を得て、1日あたりの反応数が増加
Internal Control AssayInternal ControlテンプレートQuantiFast Pathogen PCR +IC KitのInternal Control DNAテンプレートさまざまな病原体アッセイで使用できるユニバーサルDNA増幅コントロール
QuantiFast Pathogen RT-PCR +IC KitのInternal Control RNAさまざまな病原体アッセイで使用できるユニバーサルRNA増幅コントロール
Internal Control AssayMAX(HEX、VICなどに相当)で標識したプレミックスプライマー/プローブセット(TaqMan®プローブ)病原体-標的に対するプライマーに干渉しない
追加キットコンポーネントQuantiFast Pathogen RT Mix*Sensiscript Reverse Transcriptaseの独自の組成を含む病原体RNAの高感度検出用に最適化
ROX Dye SolutionApplied Biosystems 7500 real-time PCRシステムで蛍光シグナルを正規化するためのパッシブレファレンス色素の個別チューブ。オプション:AgilentのStratagene装置で使用できますROX色素を必要とするサイクラーで正確な定量。どのリアルタイムPCRサイクラーでもPCRに干渉しません
High-ROX Dye SolutionApplied Biosystems 7900とStepOne real-time PCRシステムで蛍光シグナルを正規化するためのパッシブレファレンス色素の個別チューブ
QuantiTect Nucleic Acid Dilution Buffer核酸スタンダードの希釈および保存のための独自のバッファー組成希釈および反応セットアップ中のRNAおよびDNAスタンダードを安定化し、チューブやピペットチップなどのプラスチック表面での核酸のロスを防ぎます。
図参照

操作手順

QuantiFast Pathogen +IC Kitsは、ユーザー定義の病原体とInternal Controlを検出するための簡便な手順を提供します。キットには、ウイルスRNA(1ステップRT-PCR)またはウイルス、細菌、真菌DNA(PCR)をリアルタイムで検出するための、すぐに使用できるマスターミックスが含まれています。反応条件やサイクリング条件を最適化する必要はありません。付属のマスターミックスと病原体アッセイ(プライマーとプローブ)、付属のInternal Control AssayとInternal Control DNAまたはRNAを混合するだけです。あるいは、サンプル精製手順にInternal Controlを加える場合は、Internal Control DNAまたはRNAの代わりにRNaseフリー水を反応ミックスに加えます。その後、サンプルDNAまたはRNAを加え、任意のサイクラーで反応を開始します。キットのハンドブックには、さまざまなサイクラーでTaqMan®プローブを使用することができるように最適化されたプロトコールが含まれています。また、色素の組み合わせの選択に関する推奨事項も記載されています。

各QuantiFast Pathogen +IC Kitには、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するためのInternal Control AssayとInternal Control DNAまたはRNAが含まれています。あるいは、ICを精製手順に追加して、精製プロセスと増幅の両方をコントロールすることもできます。精製手順にInternal Controlを追加するには、高濃度のInternal Control DNAまたはRNA(高濃度)を別途注文できます(図「 QIAGEN Internal Control」参照)。

図参照

アプリケーション

QuantiFast Pathogen +IC Kitsは、内部コントロールを含む病原体DNAおよび/またはRNAの検出に配列特異的プローブを使用する高感度real-time PCR または1ステップRT-PCR を提供します。このキットは、QIAGEN、Applied Biosystems、Bio-Rad、Roche(キャピラリーサイクラーを除く)、Agilentのサイクラーを含む幅広いリアルタイムPCRサイクラーで使用できます。

裏付けデータと数値

Specifications

FeaturesSpecifications
Applications病原体の検出:ウイルス、細菌、または真菌DNAのreal-time PCR(QuantiFast Pathogen PCR +IC Kit)またはウイルスRNA検出用の1ステップRT-PCR(QuantiFast Pathogen RT-PCR +IC Kit)
Sample/target typeQuantiFast Pathogen PCR +IC Kit:ウイルス、細菌、または真菌DNA;QuantiFast Pathogen RT-PCR +IC Kit:ウイルスRNA
Single or multiplexデュプレックス
Reaction type内部コントロール(IC)を含むリアルタイムPCRまたは1ステップRT-PCR
Real-time or endpointリアルタイム
SYBR Green I or sequence-specific probes配列特異的プローブ
Thermal cyclerデュプレックスPCR/RT-PCRに対応するほとんどの標準的な高速リアルタイムサイクラー(Rotor-Gene Q、またはAgilent、Applied Biosystems、BioRad、Rocheのサイクラーなど)
With or without ROXマスターミックスにはROX色素は含まれていませんが、2種類のROX溶液が含まれています:ABI 7500以外のABIサイクラーで使用するHigh-ROX Dye Solution、ABI 7500およびその他のサプライヤーで使用するROX Dye Solution(低ROX濃度)

リソース

MSDS (2)
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パンフレット (1)
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Safety Data Sheets (1)
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