本试剂盒基于耐高温逆转录酶，首先将 RNA 逆转录为 cDNA，随后由 Bst 2.0 DNA 聚合酶快速完成 DNA 扩增；同时将可视染料预混于缓冲液中，减少了加样操作步骤，扩增结束后可直接肉眼判读结果，具有极高的扩增效率和灵敏度。
组分与储存：
| 组分货号 | 组分名称 | 物理外观 | 96T | 96T×10 | 储存 | | L1540185A | Colorimetric RT-LAMP Enzyme Mix | 液体 | 200μL | 200μL×10 | -20℃ 避免反复冻融 | | L1540185B | Colorimetric RT-LAMP Reaction Buffer (2×) | 液体 | 0.7mL × 2 | 0.7mL×20 | -20℃ 避免反复冻融 | | L1540185C | 阳性对照
| 液体 | 80μL | 80μL×10 | -20℃ 避免反复冻融 | | L1540185D | 矿物油 | 液体 | 800μL × 3 | 800μL×30 | -20℃ 避免反复冻融 |
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自备材料：
1. 核心检测材料
Primer 引物：需自行设计并合成 LAMP 引物
模板 RNA：即待检测的 RNA 样本。
2. 仪器与耗材
温控设备：金属浴、水浴锅或 PCR 仪
反应管：用于配制反应的离心管/PCR 管。
移液器及配套吸头：用于精准移液。
观察辅助：光线良好的环境，以及白纸（作为背景，方便肉眼判读颜色变化）。
3. 通用试剂
ddH₂O, 用于补足反应体系。
建议反应体系：
1、将仪器设置为65°C。若使用PCR仪，将热盖温度设置为70°C。
| 组分 | 用量 | 终浓度 | | Colorimetric RT-LAMP Reaction Buffer (2 × ) | 12.5 μL | 1× | | Colorimetric RT-LAMP Enzyme Mix | 2 μL | - | | Primers (10×)* | 2.5 μL | FIP/BIP 各 1.6 μM，
LF/LB 各 0.4 μM，
F3/B3 各 0.2 μM | 模板 RNA**
| X μL | >10 copies or more | | ddH₂O | 补至 25 μL
| - |
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*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8μl P 阳性对照 （已包含Primers和模板RNA）；
注意：加入的样本避免使用Tris-HCL、PBS、TE Buffer等缓冲体系溶解或稀释，否则PH指示剂不能发生变色，影响实验结果判读。
2、65°C恒温孵育30-40min，85℃孵育5min，待冷却至室温后取出反应管，置于光线良好的环境中观察（建议以白纸作为背景）。如反应液为洋红色，则为阴性结果；如反应液为橙黄色，则为阳性结果。
结果展示：
A: ddH₂O ；B~D: 阴性样品；E~H: 每反应体系加入 5 拷贝至 5000 拷贝的 DNA 模板。 |
注意事项：
1.试剂及模板的配制操作在不同区域进行，以免发生污染。
2.建议每次实验增加无模板阴性对照。
3.温控设备可以用金属浴，水浴锅或PCR仪。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℃。
4.为防止反应液蒸发， 若使用水浴锅或金属浴，需在最终的反应体系液面上加入20 μL矿物油。若使用PCR仪，则无需加入矿物油，但必须将热盖温度设置为75°C。
5.本试剂采用pH指示剂法，需严格隔绝空气接触。长期暴露会吸附CO₂导致试剂酸化，影响检测准确性。保存条件：避免常温/冷藏，建议-20℃冷冻保存。
6.为了保证重复良好的结果，建议最后加入模板。
This kit is based on a thermostable reverse transcriptase, which first reverse-transcribes RNA into cDNA, followed by rapid DNA amplification by Bst 2.0 DNA polymerase. Meanwhile, the visual dye is pre-mixed into the buffer, reducing the number of pipetting steps. After amplification, results can be directly interpreted by the naked eye, offering extremely high amplification efficiency and sensitivity.
Contents & Storage
| Component No. | Component Name | Appearance | 96T | 96T×10 | Storage | | L1540185A | Colorimetric RT-LAMP Enzyme Mix | Liquid | 200μL | 200μL×10 | -20°C. Avoid repeated freeze-thaw cycles. | | L1540185B | Colorimetric RT-LAMP Reaction Buffer (2×) | Liquid | 0.7mL × 2 | 0.7mL×20 | -20°C. Avoid repeated freeze-thaw cycles. | | L1540185C | Positive Control | Liquid | 80μL | 80μL×10 | -20°C. Avoid repeated freeze-thaw cycles. | | L1540185D | Mineral oil | Liquid | 800μL × 3 | 800μL×30 | -20°C. Avoid repeated freeze-thaw cycles. |
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Materials Required but Not Provided:
1.Core Detection Materials
Primers: LAMP primers need to be designed and synthesized by the user.
Template RNA: The RNA sample to be tested.
2.Instruments and Consumables
Temperature control device: Metal bath, water bath, or PCR instrument.
Reaction tubes: Centrifuge tubes/PCR tubes for preparing reactions.
Pipettes and matching tips: For precise pipetting.
Observation aids: A well-lit environment and white paper (as a background for easy visual interpretation of color changes).
3.General Reagents
ddH₂O: Used to make up the final reaction volume.
Recommended Reaction System:
1.Set the instrument to 65°C. If using a PCR instrument, set the heated lid temperature to 70°C.
| Component | Volume | Final Concentration | | Colorimetric RT-LAMP Reaction Buffer (2×) | 12.5 μL | 1X | | Colorimetric RT-LAMP Enzyme Mix | 2 μL | - | | Primers (10×)* | 2.5 μL | FIP/BIP 1.6 μM each,
LF/LB 0.4 μM each,
F3/B3 0.2 μM each | | Template RNA** | × μL | >10 copies or more | | ddH₂O | Up to 25 μL | - |
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* Pipetting example (taking the preparation of a 10× Primers mix as an example):
Primer stock concentrations: Dilute FIP and BIP primers to 80 μM; dilute LF, LB, F3, and B3 primers to 20 μM.
Pipetting volumes: FIP, BIP, LF, and LB primers: 0.5 μL each; F3 and B3 primers: 0.25 μL each. (Primers may also be diluted to other concentrations; in such cases, the volumes added should be calculated accordingly based on the final concentration requirements in the reaction system.)
** For the positive control group, add 8 μL Positive Control (which already contains Primers and template RNA).
Note: Avoid dissolving or diluting the sample in buffer systems such as Tris-HCl, PBS, or TE Buffer; otherwise, the pH indicator will not change color, affecting the interpretation of experimental results.
2.Incubate at a constant temperature of 65°C for 30–40 min, then at 85°C for 5 min. After cooling to room temperature, remove the reaction tubes and observe in a well-lit environment (white paper as background is recommended). If the reaction solution is magenta, the result is negative; if the reaction solution is orange-yellow, the result is positive.
Result Display:
A: ddH₂O ; B~D: negative samples; E~H: 5 copies to 5000 copies DNA templates |
Precautions:
1.Perform the preparation of reagents and templates in separate areas to avoid contamination.
2.It is recommended to include a no-template negative control in each experiment.
3.Temperature control devices can be a metal bath, water bath, or PCR instrument. The heated lid of some PCR instrument brands defaults to 105°C after power-on. It is recommended to run a few cycles to lower the heated lid temperature to 70°C.
4.To prevent evaporation of the reaction solution: if using a water bath or metal bath, add 20 μL of mineral oil on top of the final reaction system. If using a PCR instrument, no mineral oil is needed, but the heated lid temperature must be set to 75°C.
5.This reagent uses the pH indicator method and must be strictly protected from air exposure. Long-term exposure will adsorb CO₂ and cause acidification of the reagent, affecting detection accuracy. Storage conditions: Avoid room temperature/refrigeration; storage at -20°C is recommended.
6.To ensure good reproducibility, it is recommended to add the template last.
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