RT-LAMP等温扩增试剂盒 (含荧光染料, UDG, 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。本试剂盒已将酶类与荧光染料（同 Eva green / Eve green）预混并冻干于同一管中，加水复溶后即可用于实时荧光信号监测，操作极为简便，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。
此外，本试剂盒中的 dUTP 和热敏 UDG 可有效降低残留污染风险；热敏 UDG 在温度高于 50℃ 时可完全失活，因此对后续扩增反应没有影响。
组分与储存：
| 组分货号 | 组分名称 | 物理外观 | 96T | 960T
| 储存 | | R1547589A | 反应液冻干粉* | 粉末 | 12EA | 120EA | -20℃ 避免反复冻融 | | R1547589B | UDG 酶 (1U/μL) | 液体 | 100μL | 100μL×10 | -20℃ 避免反复冻融 | | R1547589C | 阳性对照 | 液体 | 80μL | 80μL×10 | -20℃ 避免反复冻融 | | R1547589D | PCR级水 | 液体 | 1mL | 1mL×2 | -20℃ 避免反复冻融 |
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。
建议反应体系：
1、 按下表在含有反应液冻干粉的八连排中加入以下组分（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 70℃以下）。
| 组分 | 用量 | 终浓度 |
| UDG 酶 (1U/μL) | 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 80 μM；将 LF、LB、F3 与 B3 引物稀释至 20 μM。
加样体积：FIP、BIP、LF、LB引物：各 0.5 μL；F3、B3 引物：各 0.25 μL。 (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 8 μL 阳性对照 (已包含 Primers 和模板 RNA)
2、 65°C 恒温孵育 1 小时。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, UDG, Lyophilized) utilizes a system of thermostable reverse transcriptase and high-activity Bst 2.0 DNA polymerase to achieve rapid and efficient amplification of target RNA under isothermal conditions. The enzymes and fluorescent dye (similar to Eva Green / Eve Green) in this kit are pre-mixed and lyophilized in a single tube. After reconstitution with water, the product can be used for real-time fluorescence signal monitoring, featuring extremely simple operation and suitable for rapid establishment of RT-LAMP molecular detection systems.
This kit adopts 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 conformational changes and dissociates from the enzyme, releasing enzyme activity and initiating the amplification reaction. This mechanism effectively reduces non-specific amplification during reaction setup and temperature ramping, improving detection specificity, sensitivity and reproducibility.
A positive control is built into the kit, which can be used for reaction system establishment, experimental workflow verification and in-process quality control, helping users quickly complete method development and experimental setup. Featuring high amplification efficiency, high sensitivity, strong specificity and excellent system stability, this product is applicable to molecular detection, pathogenic microorganism detection, scientific research experiments and POCT application development.
In addition, the dUTP and thermolabile UDG contained in this kit can effectively reduce the risk of carryover contamination. Thermolabile UDG is completely inactivated at temperatures above 50 °C and thus has no impact on subsequent amplification reactions.
Components & Storage:
| Component No. | Component Name | Appearance | 96T | 960T
| Storage | | R1547589A | Lyophilized Reaction Mix Powder | Powder | 12EA | 120EA | -20°C. Avoid repeated freeze-thaw cycles. | | R1547589B | UDG enzyme (1U/μL) | Liquid | 100μL | 100μL×10 | -20°C. Avoid repeated freeze-thaw cycles. | | R1547589C | Positive Control | Liquid | 80μL | 80μL×10 | -20°C. Avoid repeated freeze-thaw cycles. | | R1547589D | PCR-grade Water | Liquid | 1mL | 1mL×2 | -20°C. Avoid repeated freeze-thaw cycles. |
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 to be Prepared by User:
1. Fluorescence instrument, e.g. qPCR instrument.
2. Primers, to be designed and synthesized by users.
Recommended Reaction System:
1. Prepare the reaction mixture according to the table below改为 Prepare the reaction mixture by adding the following components to the 8-well strips containing lyophilized reaction mixture according to the table below (Note: Turn off the hot lid of the PCR instrument. Make sure the hot lid is closed in advance and its temperature is controlled before running the program. For some brands of PCR instruments, the hot lid defaults to 105 °C after power-on. It is recommended to run several cycles to lower the hot lid temperature below 70 °C).
| Component | Volume | Final Concentration |
| UDG enzyme (1U/μL) | 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 | - |
*Sample loading example (for preparation of 10× Primers Mix):
Stock primer concentration: Dilute FIP and BIP primers to 80 μM; dilute LF, LB, F3 and B3 primers to 20 μM.
Loading volume: 0.5 μL each for FIP, BIP, LF and LB primers; 0.25 μL each for F3 and B3 primers. (Primers may also be diluted to other concentrations, in which case the added volume shall be calculated correspondingly according to the final concentration requirements in the reaction system.)
**Add 8 μL positive control (containing Primers and template RNA) to the positive control group.
2. Incubate isothermally at 65 °C for 1 hour. Set the qPCR instrument to read fluorescence signals once per minute (FAM channel).
Precautions:
1. Prepare reagents and perform amplification reactions in separate areas to avoid contamination.
2. A no-template negative control is recommended for each experiment.
3. This kit contains a mixture of dTTP and dUTP. Since LAMP generates massive amounts of DNA in a very short time, the best practice for contamination control is to prepare reagents and templates in separate areas and avoid opening tubes after amplification.
4. Temperature-controlled equipment can be a metal bath, water bath or PCR instrument. Turn off the hot lid of the PCR instrument. Make sure the hot lid is closed in advance and its temperature is controlled before running the program. For some brands of PCR instruments, the hot lid defaults to 105 °C after power-on. It is recommended to run several cycles to lower the hot lid temperature below 70 °C.
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