IsoFast® Bst 一步法预混液

​概述​

IsoFast® Bst 一步法预混液是一种双酶系统，用于一步法快速、灵敏地进行RNA靶标的等温扩增。

该试剂盒包含提供强大链置换能力的IsoFast® Bst DNA聚合酶，以及高活性修饰的MMLV逆转录酶RTase Go。

IsoFast® Bst 一步法预混液设计用于在单管中进行靶标RNA的逆转录及随后的等温扩增。

​特点​

• 经验证适用于SARS-CoV-2核酸的定性检测

• 包含链置换型IsoFast® Bst DNA聚合酶，呈2x预混液形式

• 提供RTase Go及RNase抑制剂

• 反应在65˚C下进行

• 对多种模板均可实现快速且稳定的扩增

• 包含先进缓冲体系，可在困难条件下获得更高产量

• 提供用于实时检测的荧光染料

• 30分钟操作流程​

​双酶系统​

该试剂盒利用具有强链置换活性的IsoFast® Bst DNA聚合酶，使其能够在恒温（65˚C）下进行DNA合成，而无需热循环。该酶代表Geobacillus stearothermophilus（原Bacillus stearothermophilus）DNA聚合酶的大片段，可催化DNA的5'-3'合成，但不含5'-3'核酸外切酶活性域¹。该酶以便捷的2x预混液形式提供。

靶标RNA的逆转录由热稳定性高且活性极强的RTase Go完成，该逆转录酶与RNase抑制剂混合，以防止污染物RNase对RNA的降解。

​快速且一致的结果​

IsoFast® Bst 一步法预混液专为快速扩增设计，可在多种样本类型中获得快速且一致的结果，并经验证适用于SARS-CoV-2核酸的定性检测（图1和图2）。其缓冲体系化学性质可确保即使在困难条件下（例如存在抑制剂时）也能获得高产量和良好性能。

IsoFast® Bst 一步法预混液包含快速、灵敏地进行RNA靶标等温扩增所需的所有组分，只需额外加入引物、模板和水即可。该试剂盒还单独提供一管荧光染料，以便与任何实时qPCR仪配合进行实时检测。

​本产品不适用于PCR。​​

¹ Mead DA, McClary JA, Luckey JA, Kostichka AJ, Witney FR, Smith LM. Bst DNA polymerase permits rapid sequence analysis from nanogram amounts of template. Biotechniques. 1991 Jul;11(1):76-8, 80, 82-87.

​应用​

• 多重置换扩增

• 等温扩增

• 逆转录环介导等温扩增

• 分子诊断

• 现场诊断

Rapid and sensitive amplification performance Isothermal amplification of beta actin from human lung total RNA using IsoFast™ Bst 1-Step Mix. A primer mix of 0.2μM for F3 and B3 primers, 1.6μM for FIP and BIP primers and 0.8μM for LoopF and LoopB primers was used and a total reaction volume of 25μL. 7 serial dilutions of template were used, corresponding to 250ng, 25ng, 2.5ng, 250pg, 25pg, 2.5pg and 250fg of total RNA. The reaction was run at 65°C for 34 minutes. A BioRad CFX96 Touch instrument was used to record fluorescence every 10 seconds. Time to result is the time required to reach the same fluorescent threshold. IsoFast™ Bst 1-Step Mix provides rapid and sensitive amplification down to 250fg of total RNA.

Rapid detection of SARS-CoV-2: Isothermal amplification of E1 target in SARS-CoV-2 RNA using IsoFast™ Bst 1-Step Mix, and compared to results obtained with NEB WarmStart® LAMP Kit. A primer mix of 0.2μM for F3 and B3, 1.6μM for FIP and BIP and 0.8μM for LoopF and LoopB primers was used. The total reaction volume was 25μL. 7 serial dilutions of template were used, corresponding to 40000, 8000, 1600, 320, 64, 12.8, 2.56 copies of SARS-CoV-2 RNA. The reaction was run at 65°C for 100 minutes. A BioRad CFX96 Touch instrument was used to record fluorescence every 10 seconds. Time to result is the time required to reach the same fluorescent threshold. IsoFast™ Bst 1-Step Mix enables rapid detection of all dilutions of SARS-CoV-2 RNA tested.

Rapid detection of SARS-CoV-2: Isothermal amplification of N2 target in SARS-CoV-2 RNA using IsoFast™ Bst 1-Step Mix, and compared to results obtained with NEB WarmStart® LAMP Kit. A primer mix of 0.2μM for F3 and B3, 1.6μM for FIP and BIP and 0.8μM for LoopF and LoopB primers was used. The total reaction volume was 25μL. 7 serial dilutions of template were used, corresponding to 40000, 8000, 1600, 320, 64, 12.8, 2.56 copies of SARS-CoV-2 RNA. The reaction was run at 65°C for 100 minutes. A BioRad CFX96 Touch instrument was used to record fluorescence every 10 seconds. Time to result is the time required to reach the same fluorescent threshold. IsoFast™ Bst 1-Step Mix enables rapid detection of all dilutions of SARS-CoV-2 RNA tested.