DNA恒溫快速擴增試劑盒(熒光型)說明書

DNA 恒溫快速擴增試劑盒（熒光型） DNA Isothermal Rapid Amplification Kit（Fluorescent Type) Guide Manual

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產品參數Product parameters：

原理概述 Principle overview：

本試劑盒基于一種常溫恒溫核酸快速擴增技術：在常溫恒溫下，重組酶和引物形成蛋白/單鏈核苷酸的復合體 Rec/ssDNA，在輔助蛋白和單鏈結合蛋白 SSB 的幫助下，侵入雙鏈 DNA 模板；在侵入位點形成 D-loop區域，并開始對 DNA 雙鏈進行掃描；待找到與引物互補的目的區域后，復合體Rec/ssDNA 解體的同時，聚合酶也結合到引物的 3’末端，開始鏈的延伸。本試劑盒在39 ºC 下，依賴核酸外切酶的作用，加入根據模板設計的特異的分子探針，使用熒光監測設備能實現對目標片段擴增過程的實時監控。

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.

引物設計 Primer design：

建議使用長度在 30-35 bp 的引物，引物過短會影響擴增速度和檢測靈敏度；引物設計避免形成二級結構而影響擴增；擴增子長度建議在 150-300 bp，通常不超過 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.

熒光探針設計Fluorescent probe design：

探針序列不與特異性引物識別位點重疊，長度為 46-52 nt，序列避免回文序列、內部二級結構和連續的重復堿基。探針共有四個修飾位點：距離 5’端的≥ 35 nt 的中部位置標記一個 dSpacer（四氫呋喃，THF），作為核酸外切酶的識別位點；THF 位點的上游標記一個熒光基團，下游標記一個粹滅基團，兩個基團的間距為2-4 nt；THF 距離3’末端≥15 nt，并且 3’末端標記一個修飾基團，例如胺基、磷酸基團或 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.

正對照熒光結果圖： P：正對照；N：陰性對照

產品特點及優勢Product features and advantages：

本試劑盒具有靈敏度高、特異性強、反應時間短（僅需 20 min）等優點，反應組分為干粉狀態，操作簡便，易于保存?？蛇m用于各種品牌的熒光定量 PCR 儀、恒溫熒光擴增儀器等熒光檢測設備。

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.

產品用途Application：

適用于實驗室級別的DNA擴增以及其他檢測用途的DNA擴增使用。

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

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