本文選題：RPA + ddPCR��； 參考：《上海海洋大學》2017年碩士論文

【摘要】：隨著轉(zhuǎn)基因技術(shù)的不斷發(fā)展與應(yīng)用,轉(zhuǎn)基因產(chǎn)品檢測技術(shù)的研究對全球轉(zhuǎn)基因產(chǎn)品安全管理至關(guān)重要。在這些檢測技術(shù)中,核酸在恒溫下的擴增技術(shù)得到了較快的發(fā)展。與常規(guī)PCR相比,核酸等溫擴增技術(shù)不再需要熱循環(huán)儀器,可在恒溫條件下快速擴增出目的片段,具有快速、簡便、靈敏的優(yōu)點。其中,重組酶聚合酶擴增技術(shù)(Recombinase ploymerase amplification,RPA),是核酸在等溫條件下進行擴增的一種技術(shù)。微滴數(shù)字PCR(droplet digital PCR,ddPCR),是一種核酸檢測與定量技術(shù),也是最近幾年興起的新技術(shù)。本研究的內(nèi)容和結(jié)果如下:1、RPA結(jié)合凝膠檢測技術(shù)特異性檢測轉(zhuǎn)基因玉米Bt11:本研究根據(jù)轉(zhuǎn)基因玉米Bt11外源基因草丁膦乙酰轉(zhuǎn)移酶基因(phosphinothricin acetyl transferase gene,PAT)及載體骨架連接區(qū)序列,設(shè)計引物,首次建立了基于RPA與凝膠檢測技術(shù)相結(jié)合的方法,特異性地快速檢測轉(zhuǎn)基因玉米Bt11,在37℃條件下20min內(nèi)即可完成檢測。本實驗所得該方法的絕對檢測限約為100拷貝,相對檢測限約為0.1%。2、RPA結(jié)合熒光檢測技術(shù)檢測轉(zhuǎn)基因玉米Bt11外源基因nos終止子:此研究選用轉(zhuǎn)基因玉米Bt11為材料,研究RPA熒光技術(shù)檢測外源基因nos終止子的靈敏度,測得絕對和相對檢測限分別約為50拷貝和0.1%。與之對比實驗,本實驗采用熒光定量PCR染料法檢測轉(zhuǎn)基因玉米Bt11外源基因nos終止子,實驗測得絕對檢測限達10拷貝甚至以下,相對檢測限為0.1%。熒光定量PCR可對轉(zhuǎn)基因玉米含量進行定量測定,但熒光定量PCR相對檢測時間較長,需要兩小時以上,并且定量時需要做標準曲線,過程復雜,而應(yīng)用RPA熒光技術(shù)檢測時間明顯縮短,半小時就可以完成檢測。兩種技術(shù)各有優(yōu)勢,可根據(jù)實際需要選取。3、ddPCR測定轉(zhuǎn)基因大豆GTS-40-3-2轉(zhuǎn)基因成分含量:在本研究中,使用了ddPCR和熒光定量PCR對10%的轉(zhuǎn)基因大豆GTS-40-3-2標準品進行了轉(zhuǎn)基因成分含量的測定,兩種方法測得轉(zhuǎn)基因含量與實際相符,都約為10%。相比ddPCR方法,熒光定量PCR需要更多的模板量和引物,加樣的數(shù)量也比較多。采用QX200TM Droplet Digital TM PCR系統(tǒng),將兩套引物和探針加入到同一PCR管中,減少了加樣數(shù)量,也節(jié)省了模板量。在數(shù)據(jù)分析方面,熒光定量PCR方法需要根據(jù)熒光數(shù)值構(gòu)建標準曲線,并通過計算獲得基因的拷貝數(shù)和轉(zhuǎn)基因成分的含量,而ddPCR不依賴標準曲線,能夠通過不同熒光的微滴數(shù)目直接獲得靶基因的拷貝數(shù),并精確計算出轉(zhuǎn)基因成分所占的百分數(shù)。本實驗所得ddPCR方法的最低檢出限約為2個拷貝。
[Abstract]:With the development and application of transgenic technology, the research of transgenic product detection technology is very important to the global safety management of transgenic products.Among these detection techniques, nucleic acid amplification at constant temperature has been developed rapidly.Compared with conventional PCR, the nucleic acid isothermal amplification technique no longer requires a thermal cycle instrument, and can rapidly amplify the target fragment under the condition of constant temperature, which has the advantages of rapidity, simplicity and sensitivity.Among them, Recombinase ploymerase amplification is a technique for nucleic acid amplification under isothermal conditions.Microdrop digital PCR(droplet digital rcad PCR is a nucleic acid detection and quantification technique, and it is also a new technology developed in recent years.The contents and results of this study were as follows: Bt11 of transgenic maize was specifically detected by the combination of 1: 1 RPA and gel detection. Primers were designed based on phosphinothricin acetyl transferase gene (PATT) and the sequence of vector skeleton junction region (PATs) of transgenic maize Bt11.For the first time, a method based on RPA and gel detection was established to detect transgenic maize BT 11 specifically and quickly, which could be detected in 20min at 37 鈩，

本文編號：1774128