S_1084_5_GEN_V2

✓ 24/7 automatic processing of online orders

✓ Knowledgeable and professional Product & Technical Support

✓ Fast and reliable (re)-ordering

QuantiTect Virus Kit (1000)

Cat. No. / ID:  211015

For 1000 x 50 µl reactions: QuantiTect Virus Master Mix (contains ROX dye), QuantiTect Virus RT Mix, RNase-Free Water, QuantiTect Nucleic Acid Dilution Buffer
Copy order details
$4,888.00
Log in To see your account pricing.
Reference dye
Rox in Mastermix
Rox in vial
Reactions
1000
200
50
QuantiTect Virus 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。

✓ 24/7 automatic processing of online orders

✓ Knowledgeable and professional Product & Technical Support

✓ Fast and reliable (re)-ordering

Features

  • シングルアッセイだけでなくマルチプレックスアッセイでも高感度を実現
  • 同一反応液中でウイルスRNA/DNAの検出が可能
  • 低い陽性シグナルも明確に検出
  • 迅速でユニバーサルな2ステッププロトコール
  • 5x マスターミックスでサンプル添加量を増量でき感度を増大

Product Details

QuantiTect Virus Kit は配列特異的なプローブを用いた1ステップマルチプレックスRT-PCRにより、最高4種類のウイルス核酸を高感度に検出するため特別にデザインされました。マルチプレックスアッセイでも、感度を損なうことなく複数のウイルスRNA/DNA とインターナルコントロールを検出できます。添付の5x マスターミックスにはHotStarTaq Plus DNA Polymeraseが含まれています。濃縮されているので、添加するサンプルの量を増やせます。QuantiTect Virus RT Mix は、ウイルスRNAの高感度な検出のために至適化されたSensiscript Reverse Transcriptaseをユニークな配合で含んでいます。2種類のキットフォーマットが入手可能です(蛍光補正のためにROX色素が必要なサイクラー用のQuantiTect Virus Kitとその他のサイクラー用のQuantiTect Virus +ROX Vial Kit)。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。

Performance

QuantiTect Virus Kitsを用いて増幅すると、希釈範囲にわたってシャープなS字曲線を示し、テンプレートが少量でも高いCT 値をとります(図 " 幅広いダイナミックレンジで明確なCT 値を測定可能")。その結果、リアルタイムPCR におけるウイルス核酸定量で正確な CT 値の決定が可能です。

マルチプレックスアッセイにより、感度を損なうことなく複数のウイルスRNAやDNA ターゲットおよびインターナルコントロールの幅広い範囲で直線性のある検出が可能です(図 " 幅広い範囲で直線性のあるウイルスRNA 検出"および" 従来のマルチプレックスよりも低濃度でウイルスRNAを検出")。

キットに同梱のQuantiTect Nucleic Acid Dilution Buffer は、希釈中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。このバッファーは、ウイルス核酸定量のためのスタンダード用検体の信頼できる希釈を可能にし、CT 値が低値から高値まで幅広い範囲で直線性のある測定を実現するとともに、スタンダード用検体の劣化のない長期保存を保証します(図 " RNAスタンダードの正確な希釈および保存")。

See figures

Principle

QuantiTect Virus Kit は、シングル/マルチプレックスアッセイあるいは1ステップRT-PCRで初回実験からウイルス核酸を高感度に検出します(フローチャート" QIAGEN multiplex kits)。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PCR反応産物は、対応するシングルPCR反応産物と同じ効率と感度で増幅されます。

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、手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。QuantiTect Virus Bufferに入っている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の独自の配合により、ウイルスRNAの高感度逆転写を実現し、また、HotStarTaq Plus DNA Polymeraseにより厳密なホットスタートが可能になり、非特異的産物の形成を防ぎます。

QuantiTect Probe RT-PCR Kit の成分
成分 特長 利点
5x QuantiTect Virus Master Mix 濃縮マスターミックス 高感度な病原体検出用に高濃縮かつ至適化済み 高濃度マスターミックスのためテンプレート量を増やし感度を高めることが可能
HotStarTaq Plus DNA Polymerase 95℃、5分の活性化 室温での定量PCRのセットアップ
QuantiTect Virus Buffer NH4+イオンおよび 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 同一チューブで4遺伝子まで信頼できるマルチプレックス解析が可能
付属のキット構成 QuantiTect Virus RT Mix ユニークな組成のSensiscript Reverse Transcriptase ウイルスRNAの高感度検出用に至適化済み
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 QuantiTect Nucleic Acid Dilution Bufferは、反応セットアップ中における標準曲線用の連続希釈RNA/DNAサンプルを安定化し、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ぐ
See figures

Procedure

QuantiTect Virus Kitsは、配列特異的プローブを用いたウイルス核酸(RNA/DNA)および内部コントロールの高感度なリアルタイムPCR解析を実現します。逆転写反応「あり」「なし」のどちらも可能であり、標的のRNA、DNA、あるいはRNAおよびDNAの両方を検出するマルチプレックスアッセイを柔軟に設計できます。ハンドブックに記載されているプロトコールに従えば、迅速で正確な結果が得られます。

マスターミックス中にROX passive reference dyeが入ったキットまたは入っていないキットをお求めいただけます(表を参照)。

QuantiTect Multiplex PCR Kitの選択ガイド
ROX 色素 キット 対応するサイクラー
マスターミックスに添加済み QuantiTect Virus Kit Applied Biosystems 7500以外のApplied Biosystemsの全てのサイクラー
別チューブで添付 QuantiTect Virus +ROX Vial Kit Applied Biosystems 7500 およびBio-Rad、Cepheid、Eppendorf、QIAGEN、Roche、Agilent、その他の会社のサイクラー

ウイルス検出などの用途での迅速で高感度な1ステップのエンドポイントRT-PCRアプリケーション用には、QIAGEN OneStep RT-PCR Kit を推奨します。

Applications

QuantiTect Virus Kit は、ウイルスDNA/RNAおよびインターナルコントロール検出のために配列特異的なプローブを用いて高感度なシングル/マルチプレックスのリアルタイムPCRあるいは1ステップリアルタイムRT-PCRを実現します。本キットはApplied Biosystems、Bio-Rad、Cepheid、Eppendorf、QIAGEN、Roche(キャピラリー式サイクラーを除く)、Agilent社製を含む様々なリアルタイムサイクラー上で使用できます。

Supporting data and figures

Specifications

FeaturesSpecifications
ApplicationsVirus detection
SYBR Green I or sequence-specific probesSequence-specific probes
Real-time or endpointReal-Time
Reaction typeReverse Transcription and PCR
Thermal cyclerMost real-time cyclers (except capillary cyclers e.g. LightCycler® 1.x and 2.0)
Sample/target typeRNA and/or DNA targets
With or without ROXAvailable with ROX in master mix and ROX as a separate vial
Single or multiplexSingle or multiplex

Resources

パンフレット (1)
Now with even more applications!
MSDS (1)
Download Safety Data Sheets for QIAGEN product components.
キットハンドブック (1)
For highly sensitive real-time singleplex or multiplex PCR or one-step RT-PCR using sequence-specific probes for detection of viral DNA and RNA and internal controls
Certificates of Analysis (1)
Brochures & Guides (1)
Now with even more applications!
Kit Handbooks (1)
For highly sensitive real-time singleplex or multiplex PCR or one-step RT-PCR using sequence-specific probes for detection of viral DNA and RNA and internal controls

FAQ

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
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
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
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
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 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
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
Why should DNA or cDNA targets be less than 250 bp long for real-time PCR?

Shorter amplification products facilitate high PCR efficiencies. Ideally, amplicon length should be less than 150 bp for optimal amplification efficiency. PCR efficiencies close to 100% are a crucial prerequisite for accurate quantification of target copy numbers in real-time PCR.

FAQ ID-751
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
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
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