DNA 恒温快速扩增试剂盒（荧光型） DNA Isothermal Rapid Amplification Kit（Fluorescent Type) Guide Manual

产品参数Product parameters：

原理概述 Principle overview：

本试剂盒基于一种常温恒温核酸快速扩增技术：在常温恒温下，重组酶和引物形成蛋白/单链核苷酸的复合体 Rec/ssDNA，在辅助蛋白和单链结合蛋白 SSB 的帮助下，侵入双链 DNA 模板；在侵入位点形成 D-loop区域，并开始对 DNA 双链进行扫描；待找到与引物互补的目的区域后，复合体Rec/ssDNA 解体的同时，聚合酶也结合到引物的 3’末端，开始链的延伸。本试剂盒在39 ºC 下，依赖核酸外切酶的作用，加入根据模板设计的特异的分子探针，使用荧光监测设备能实现对目标片段扩增过程的实时监控。

This kit is based on a room temperature and constant temperature nucleic acid rapid amplification technology: at room temperature and constant temperature, the recombinase and primer form the protein/single-stranded nucleotide complex Rec/ssDNA, with the help of auxiliary proteins and single-stranded binding protein SSB , invade the double-stranded DNA template; form a D-loop region at the invasion site, and start scanning the DNA double-strands; after finding the target region complementary to the primer, the Rec/ssDNA complex disintegrates, and the polymerase also binds to The 3' end of the primer initiates chain extension. This kit relies on the action of exonuclease at 39 ºC, adding specific molecular probes designed based on the template, and using fluorescence monitoring equipment to achieve real-time monitoring of the amplification process of the target fragment.

引物设计 Primer design：

建议使用长度在 30-35 bp 的引物，引物过短会影响扩增速度和检测灵敏度；引物设计避免形成二级结构而影响扩增；扩增子长度建议在 150-300 bp，通常不超过 500 bp。

It is recommended to use primers with a length of 30-35 bp. Primers that are too short will affect amplification speed and detection sensitivity; primers are designed to avoid the formation of secondary structures that affect amplification; the amplicon length is recommended to be 150-300 bp, usually no more than 500 bp.

荧光探针设计Fluorescent probe design：

探针序列不与特异性引物识别位点重叠，长度为 46-52 nt，序列避免回文序列、内部二级结构和连续的重复碱基。探针共有四个修饰位点：距离 5’端的≥ 35 nt 的中部位置标记一个 dSpacer（四氢呋喃，THF），作为核酸外切酶的识别位点；THF 位点的上游标记一个荧光基团，下游标记一个粹灭基团，两个基团的间距为2-4 nt；THF 距离3’末端≥15 nt，并且 3’末端标记一个修饰基团，例如胺基、磷酸基团或 C3-Spacer。

The probe sequence does not overlap with the specific primer recognition site, is 46-52 nt in length, and the sequence avoids palindromic sequences, internal secondary structures, and continuous repeated bases. The probe has four modification sites: the middle position ≥ 35 nt from the 5' end is labeled with a dSpacer (tetrahydrofuran, THF) as the recognition site for exonuclease; the upstream of the THF site is labeled with a fluorescent group, and the downstream Label a quenching group, the distance between the two groups is 2-4 nt; THF is ≥15 nt from the 3' end, and the 3' end is labeled with a modifying group, such as an amine group, a phosphate group or a C3-Spacer.

正对照荧光结果图： P：正对照；N：阴性对照

产品特点及优势Product features and advantages：

本试剂盒具有灵敏度高、特异性强、反应时间短（仅需 20 min）等优点，反应组分为干粉状态，操作简便，易于保存。可适用于各种品牌的荧光定量 PCR 仪、恒温荧光扩增仪器等荧光检测设备。

This kit has the advantages of high sensitivity, strong specificity, and short reaction time (only 20 minutes). The reaction components are in dry powder state, which is easy to operate and easy to store. It can be applied to various brands of fluorescence quantitative PCR instruments, constant temperature fluorescence amplification instruments and other fluorescence detection equipment.

产品用途Application：

适用于实验室级别的DNA扩增以及其他检测用途的DNA扩增使用。

Suitable for laboratory-grade DNA amplification and DNA amplification for other detection purposes.

操作步骤 Operation procedure and process：

实验操作相关视频，请微信扫码联系K8凯发国际生物客服获取。

For videos related to experimental operations, please scan the QR code on WeChat to contact customer service.

产品线Product Line：

性能对比数据Performance comparison data：

Q：如何避免实验失败？

A：使用前请仔细阅读说明书，严格按照操作步骤进行操作；

Q：实验不规范会影响结果准确度么？

A：会的，样品处理应该严格按照生物安全规范操作，在进行反应时请注意避免核酸污染，并设置空白对照；

Q：试剂的存储条件？

A：储存条件：储存温度 ≤ -20 °C恒温环境，避光保存，避免重压；