RT-LAMP等温扩增试剂盒 (含荧光染料, 冻干版)基于耐热型逆转录酶和高活性的Bst 2.0 DNA聚合酶体系，可在恒温条件下实现目标RNA的快速、高效扩增。试剂盒内含荧光染料，可用于实时荧光信号监测，操作简便，适用于快速建立RT-LAMP分子检测体系。
本试剂盒采用适配体（Aptamer）介导的热启动技术。在低温条件下，适配体可与Bst DNA聚合酶特异性结合，抑制其聚合酶活性；当反应升温至工作温度后，适配体构象发生变化并从酶上解离，从而释放酶活性并启动扩增反应。该机制可有效减少反应体系配制及升温阶段的非特异性扩增，提高检测的特异性、灵敏度和重复性。
试剂盒内置阳性对照，可用于反应体系建立、实验流程验证及过程质量控制，帮助用户快速完成方法开发与实验搭建。产品具有扩增效率高、灵敏度高、特异性强及体系稳定性好等特点，适用于分子检测、病原微生物检测、科研实验及POCT应用开发等场景。
组分与储存：
| 组分货号 | 组分名称 | 物理外观 | 96T | 96T×10
| 储存 | | R1547530A | RT-LAMP Enzyme Mix(25×)-冻干粉* | 粉末 | 96T | 96T×10
| -20℃ 避免反复冻融 | | R1547530B | 复溶液 (2×) | 液体 | 0.7mL× 2 | 0.7mL×20
| -20℃ 避免反复冻融 | | R1547530C | 荧光染料 (25×) | 液体 | 100μL | 100μL×10
| -20℃ 避免反复冻融 | | R1547530D | 阳性对照 | 液体 | 80μL | 80μL×10
| -20℃ 避免反复冻融 | | R1547530E | PCR级水 | 液体 | 1mL | 1mL×10
| -20℃ 避免反复冻融 |
* 使用前请按照管身标签所示加入 PCR级水。
注意：开盖前，请先以 3000–4000 rpm 短暂离心 1 分钟，使试剂充分收集至管底。 |
自备材料：
1.荧光仪，例如Q-PCR仪。
2.Primer引物，需自行设计合成。
配制反应体系：
1、按下表配制反应混合液（注：PCR仪确保关闭热盖，务必提前关闭热盖后运行控制热盖温度。某些品牌PCR仪开机后热盖会默认升温到105℃。建议运行几个循环，将热盖温度降至75℃以下）：
| 组分 | 用量 | 终浓度 |
| 复溶液 (2×) | 12.5 μL | 1× |
| RT-LAMP Enzyme Mix (25×) | 1 μL | 1× |
| 荧光染料 (25×) | 1 μL | 1× |
| Primers (10×)* | 2.5 μL | 1.6 μM FIP/BIP, 0.4 μM LF/LB, 0.2 μM F3/B3 |
| 模板 RNA** | 1 μL | >10 copies or more |
| PCR级水 | Up to 25 μL | - |
*加样示例（以配制10× Primers混合液为例）：
引物储备液浓度：将FIP与BIP引物稀释至80μM；将LF、LB、F3与B3引物稀释至20μM。
加样体积：FIP、BIP、LF、LB引物：各0.5μL；F3、B3引物：各0.25μL。（引物亦可稀释为其他浓度，此时添加体积需根据反应体系中的终浓度要求进行相应计算。）
**阳性对照组加入8μl 阳性对照 (已包含Primers和模板RNA)
2、65℃恒温孵育1h。设置荧光定量PCR仪每分钟读取一次荧光信号。(FAM荧光通道)
注意事项：
1.试剂配制与试剂反应在不同区域进行，以免发生污染。
2.建议每次实验增加无模板阴性对照。
3.该试剂盒中含有dTTP与dUTP的混合物；由于LAMP能在极短时间内产生大量DNA，减少污染的最佳操作为试剂及模板的配制操作在不同区域进行，并且扩增后避免开盖操作。
4.温控设备可以用金属浴，水浴锅或PCR仪。PCR仪确保关闭热盖，务必提前关闭热盖后运行控制热盖温度。某些品牌PCR仪开机后热盖会默认升温到105℃。建议运行几个循环，将热盖温度降至70℃以下。
RT-LAMP Isothermal Amplification Kit (with Fluorescent Dye, Lyophilized) is based on a thermostable reverse transcriptase and a highly active Bst 2.0 DNA polymerase system, which can achieve rapid and highly efficient amplification of target RNA under isothermal conditions. The kit contains a fluorescent dye for real-time fluorescent signal monitoring. It is easy to operate and suitable for the rapid establishment of RT-LAMP molecular detection systems.
This kit utilizes an aptamer-mediated hot-start technology. At low temperatures, the aptamer specifically binds to Bst DNA polymerase and inhibits its polymerase activity; when the reaction is heated to the working temperature, the aptamer undergoes a conformational change and dissociates from the enzyme, thereby releasing the enzyme activity and initiating the amplification reaction. This mechanism can effectively reduce non-specific amplification during the reaction setup and heating stages, improving detection specificity, sensitivity, and reproducibility.
The kit includes an Positive Control, which can be used for reaction system establishment, experimental workflow verification, and process quality control, helping users quickly complete method development and experimental setup. The product features high amplification efficiency, high sensitivity, strong specificity, and good system stability, making it suitable for scenarios such as molecular detection, pathogenic microorganism detection, scientific research experiments, and POCT application development.
Contents & Storage
| Component No. | Component Name | Appearance | 96T | 96T×10 | Storage | | R1547530A | RT-LAMP Enzyme Mix (25X)-Lyophilized Powder* | Powder | 96T | 96T×10 | -20°C. H180 | | R1547530B | Reconstitution Solution (2×) | Liquid | 0.7mL× 2 | 0.7mL×20
| -20°C. Avoid repeated freeze-thaw cycles. | | R1547530C | Fluorescent Dye (25×) | Liquid | 100μL | 100μL×10 | -20°C. Avoid repeated freeze-thaw cycles. | | R1547530D | Positive Control | Liquid | 80μL | 80μL×10 | -20°C. Avoid repeated freeze-thaw cycles. | | R1547530E | PCR-grade Water | Liquid | 1mL | 1mL×10 | -20°C. Avoid repeated freeze-thaw cycles. |
*Prior to use, add PCR-grade water as indicated on the tube label.
Note: Before opening the cap, briefly centrifuge at 3000–4000 rpm for 1 min to collect all reagents at the bottom of the tube. |
Materials Required but Not Provided:
1.Fluorescence instrument, such as a Q-PCR instrument.
2.Primers, which need to be designed and synthesized independently.
Preparation of Reaction System:
1.Prepare the reaction mixture according to the table below (Note: Ensure the heated lid of the PCR instrument is turned off. Be sure to turn off the heated lid in advance before running and control the heated lid temperature. The heated lid of some brands of PCR instruments defaults to heating up to 105°C after startup. It is recommended to run a few cycles to lower the heated lid temperature below 75°C):
| Component | Volume | Final Concentration |
| Reconstitution Solution (2×) | 12.5 μL | 1×
|
| RT-LAMP Enzyme Mix (25×) | 1 μL | 1×
|
| Fluorescent Dye (25×) | 1 μL | 1×
|
| Primers (10×)* | 2.5 μL | 1.6 μM FIP/BIP, 0.4 μM LF/LB, 0.2 μM F3/B3
|
| Template RNA** | 1 μL | >10 copies or more |
| PCR-grade water | Up to 25 μL | - |
*Example of adding samples (using the preparation of 10× Primers mixture as an example):
Primer stock solution concentrations: Dilute FIP and BIP primers to 80 μM; dilute LF, LB, F3, and B3 primers to 20 μM.
Addition volumes: FIP, BIP, LF, and LB primers: 0.5 μL each; F3 and B3 primers: 0.25 μL each. (Primers can also be diluted to other concentrations; in this case, the added volume needs to be calculated accordingly based on the final concentration requirements in the reaction system.)
**For the positive control group, add 8 μl of Positive Control (which already contains Primers and template RNA).
2.Incubate at a constant temperature of 65°C for 1 h. Set the real-time PCR instrument to read the fluorescent signal once every minute. (FAM fluorescence channel)
Precautions:
1.Reagent preparation and reagent reactions should be performed in different areas to avoid contamination.
2.It is recommended to include a no-template negative control in each experiment.
3.The kit contains a mixture of dTTP and dUTP; since LAMP can produce a large amount of DNA in a very short time, the best operation to reduce contamination is to perform reagent and template preparation in different areas, and avoid opening the tubes after amplification.
4.Temperature control equipment can be a metal bath, water bath, or PCR instrument. For a PCR instrument, ensure the heated lid is turned off; be sure to turn off the heated lid in advance before running and control the heated lid temperature. The heated lid of some brands of PCR instruments defaults to heating up to 105°C after startup. It is recommended to run a few cycles to lower the heated lid temperature below 70°C.
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