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
- 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
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。
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QuantiFast Pathogen PCR +IC Kit (100)
For 100 x 25 µl reactions: Master Mix, lyophilized Internal Control Assay, lyophilized Internal Control DNA, ROX Dye Solution, High-ROX Dye Solution, RNase-Free Water, Nucleic Acid Dilution Buffer, Buffer TE
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211352
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QuantiFast Pathogen PCR +IC Kit (400)
For 400 x 25 µl reactions: Master Mix, lyophilized Internal Control Assay, lyophilized Internal Control DNA, ROX Dye Solution, High-ROX Dye Solution, RNase-Free Water, Nucleic Acid Dilution Buffer, Buffer TE
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211354
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QuantiFast Pathogen RT-PCR +IC Kit (100)
For 100 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
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211452
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QuantiFast Pathogen RT-PCR +IC Kit (400)
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
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211454
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Internal Control DNA (High conc.)
For approximately 200 preps (depending on the elution volume): Lyophilized Internal Control DNA, Nucleic Acid Dilution Buffer
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211392
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Internal Control RNA (High conc.)
For approximately 200 preps (depending on elution volume): Lyophilized Internal Control RNA, Nucleic Acid Dilution Buffer
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211492
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QuantiFast Pathogen +IC Kits are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.
Correct interpretation of negative results.|Sensitive detection of BHV-1 on the Rotor-Gene Q.|Sensitive detection of Norovirus.|Sensitive detection of BHV-1 on the ABI 7500.|Fast primer annealing.|QIAGEN multiplex kits.|Unique PCR buffer.|QIAGEN Internal Control.|High linearity and precision of singleplex and duplex detection.|Reliable dilution and storage of RNA standards.|
Duplicates of two concentrations of a viral RNA target were co-amplified with the Internal Control in the presence of different amounts of a PCR inhibitory substance (humic acid) on the Rotor-Gene Q. [A] No template controls (NTCs) serve as a reference for Internal Control signal. [B] Amplification of viral RNA target and Internal Control confirms successful amplification. [C] The Internal Control indicates the presence of a low amount of inhibitors. [D] Failure to detect the Internal Control shows the failure of the amplification reaction through presence of inhibitors.|Bovine herpes virus type 1 was serially diluted (100 to 10-4) and detected by either singleplex real-time PCR or by duplex real-time PCR in parallel with the Internal Control. Real-time PCR was carried out using the QuantiFast Pathogen PCR +IC Kit on the Rotor-Gene Q according to the supplied protocols and without any PCR optimization. The duplex reactions contained a fixed amount of Internal Control template. Each dilution was analyzed in triplicate; one replicate is shown for each dilution.|A Norovirus RNA transcript was serially diluted (100 to 10-5) and detected by either singleplex real-time RT-PCR or by duplex real-time PCR in parallel with the Internal Control. Real-time PCR was carried out using the QuantiFast Pathogen RT-PCR +IC Kit on the Rotor-Gene Q without any PCR optimization. Duplex reactions contained a fixed amount of Internal Control template. Each dilution was analyzed in triplicate; one replicate per dilution is shown.|Bovine herpes virus type 1 was serially diluted (100 to 10-4) and detected by either singleplex real-time PCR or by duplex real-time PCR in parallel with the Internal Control. Real-time PCR was carried out using the QuantiFast Pathogen PCR +IC Kit on the ABI 7500 according to the supplied protocols and without any PCR optimization. The duplex reactions contained a fixed amount of Internal Control template. Each dilution was analyzed in triplicate; one replicate is shown for each dilution.|[A] Q-Bond in QuantiFast Buffer allows the DNA polymerase and primer to bind as a single complex, reducing the annealing time to a few seconds. In addition, the unique buffer composition supports the melting of DNA, reducing denaturation and extension times. [B] Without Q-Bond, the primer and polymerase bind sequentially to the template, increasing annealing time.|QuantiFast Pathogen +IC Kits provide a simple procedure for quantitative, multiplex, real-time PCR. In contrast to current methods, the kits eliminate the need for optimization of the concentrations of primers, Mg2+, and Taq DNA polymerase, even for difficult assays (e.g., assays in which the copy number of the target gene is much smaller than that for the reference gene).|[A] NH4+ ions prevent nonspecific primers from annealing to the template. [B] Synthetic Factor MP, an innovative PCR additive, increases the local concentration of primers at the template. Together with K+ and other cations, Factor MP stabilizes specifically bound primers, allowing efficient primer extension by HotStarTaq Plus DNA Polymerase.|Simultaneous extraction and/or amplification of a pathogen target plus an internal positive control will rule out PCR inhibition or other problems that could give a false-negative result, leading to higher process safety. The QIAGEN Internal Control can be added prior to PCR amplification to provide an amplification control, or highly concentrated QIAGEN Internal Control can be added during nucleic acid extraction to provide both an extraction and an amplification control.|A 6-log range of both Norovirus RNA singleplex detection and Norovirus/IC duplex detection shows high precision and linearity. Error bars each represent ±1 SD of 3 real-time RT-PCR replicates.|Serial tenfold dilutions of RNA standards (in vitro transcribed RNA) were prepared using either QuantiTect Nucleic Acid Dilution Buffer or RNase-free water, as indicated. These dilutions were used as template in one-step RT-PCR either directly (0 test) or after storage for 2 or 4 weeks at -20°C. Using QuantiTect Nucleic Acid Dilution Buffer resulted in lower CT values and improved stability of the standards.|
Principle
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。
| 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 |
Procedure
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”)。
Applications
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。
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Images
Correct interpretation of negative results.
Duplicates of two concentrations of a viral RNA target were co-amplified with the Internal Control in the presence of different amounts of a PCR inhibitory substance (humic acid) on the Rotor-Gene Q. [A] No template controls (NTCs) serve as a reference for Internal Control signal. [B] Amplification of viral RNA target and Internal Control confirms successful amplification. [C] The Internal Control indicates the presence of a low amount of inhibitors. [D] Failure to detect the Internal Control shows the failure of the amplification reaction through presence of inhibitors.
Sensitive detection of BHV-1 on the Rotor-Gene Q.
Bovine herpes virus type 1 was serially diluted (100 to 10-4) and detected by either singleplex real-time PCR or by duplex real-time PCR in parallel with the Internal Control. Real-time PCR was carried out using the QuantiFast Pathogen PCR +IC Kit on the Rotor-Gene Q according to the supplied protocols and without any PCR optimization. The duplex reactions contained a fixed amount of Internal Control template. Each dilution was analyzed in triplicate; one replicate is shown for each dilution.
Sensitive detection of Norovirus.
A Norovirus RNA transcript was serially diluted (100 to 10-5) and detected by either singleplex real-time RT-PCR or by duplex real-time PCR in parallel with the Internal Control. Real-time PCR was carried out using the QuantiFast Pathogen RT-PCR +IC Kit on the Rotor-Gene Q without any PCR optimization. Duplex reactions contained a fixed amount of Internal Control template. Each dilution was analyzed in triplicate; one replicate per dilution is shown.
Sensitive detection of BHV-1 on the ABI 7500.
Bovine herpes virus type 1 was serially diluted (100 to 10-4) and detected by either singleplex real-time PCR or by duplex real-time PCR in parallel with the Internal Control. Real-time PCR was carried out using the QuantiFast Pathogen PCR +IC Kit on the ABI 7500 according to the supplied protocols and without any PCR optimization. The duplex reactions contained a fixed amount of Internal Control template. Each dilution was analyzed in triplicate; one replicate is shown for each dilution.
Fast primer annealing.
[A] Q-Bond in QuantiFast Buffer allows the DNA polymerase and primer to bind as a single complex, reducing the annealing time to a few seconds. In addition, the unique buffer composition supports the melting of DNA, reducing denaturation and extension times. [B] Without Q-Bond, the primer and polymerase bind sequentially to the template, increasing annealing time.
QIAGEN multiplex kits.
QuantiFast Pathogen +IC Kits provide a simple procedure for quantitative, multiplex, real-time PCR. In contrast to current methods, the kits eliminate the need for optimization of the concentrations of primers, Mg2+, and Taq DNA polymerase, even for difficult assays (e.g., assays in which the copy number of the target gene is much smaller than that for the reference gene).
Unique PCR buffer.
[A] NH4+ ions prevent nonspecific primers from annealing to the template. [B] Synthetic Factor MP, an innovative PCR additive, increases the local concentration of primers at the template. Together with K+ and other cations, Factor MP stabilizes specifically bound primers, allowing efficient primer extension by HotStarTaq Plus DNA Polymerase.
QIAGEN Internal Control.
Simultaneous extraction and/or amplification of a pathogen target plus an internal positive control will rule out PCR inhibition or other problems that could give a false-negative result, leading to higher process safety. The QIAGEN Internal Control can be added prior to PCR amplification to provide an amplification control, or highly concentrated QIAGEN Internal Control can be added during nucleic acid extraction to provide both an extraction and an amplification control.
High linearity and precision of singleplex and duplex detection.
A 6-log range of both Norovirus RNA singleplex detection and Norovirus/IC duplex detection shows high precision and linearity. Error bars each represent ±1 SD of 3 real-time RT-PCR replicates.
Reliable dilution and storage of RNA standards.
Serial tenfold dilutions of RNA standards (in vitro transcribed RNA) were prepared using either QuantiTect Nucleic Acid Dilution Buffer or RNase-free water, as indicated. These dilutions were used as template in one-step RT-PCR either directly (0 test) or after storage for 2 or 4 weeks at -20°C. Using QuantiTect Nucleic Acid Dilution Buffer resulted in lower CT values and improved stability of the standards.
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