本文選題：轉(zhuǎn)基因 + 恒溫擴增��； 參考：《浙江大學》2016年博士論文

【摘要】：轉(zhuǎn)基因作物的推廣和商業(yè)化種植在帶給人們巨大經(jīng)濟效益的同時,其潛在風險卻也飽受爭議,快速準確地檢測轉(zhuǎn)基因具有十分重要的意義,事關(guān)國際政治和經(jīng)濟利益。傳統(tǒng)基于外源蛋白分析的檢測方法具有明顯的局限性,而基于外源核酸分析的檢測方法在對操作人員有較高要求的同時又受制于專業(yè)、昂貴、體積龐大的儀器設(shè)備。為了開發(fā)簡單、快速、便攜基于核酸分析的轉(zhuǎn)基因檢測方法,本課題以抗蟲轉(zhuǎn)基因水稻華恢1號、科豐6號和克螟稻1號為研究材料,以交叉引物恒溫擴增技術(shù)(CPA)和環(huán)介導恒溫擴增技術(shù)(LAMP)為擴增方法,以核酸擴增過程中的副產(chǎn)物H+和磷酸根為檢測對象,分別建立了電化學無標記的核酸擴增檢測方法和新型可視化核酸恒溫擴增方法,并在以上研究基礎(chǔ)上提出了基于邏輯判斷的轉(zhuǎn)基因具體品系的快速鑒定策略,開發(fā)出紙上可視化點陣列鑒定轉(zhuǎn)基因品系的方法。本文的主要內(nèi)容和研究結(jié)果如下：1)針對轉(zhuǎn)基因通用元件T-nos的基因序列建立了用于轉(zhuǎn)基因作物快速篩選的CPA體系,設(shè)計了6條引物,包括2條外引物、2條交叉引物和2條檢測引物,特異性識別'T-nos序列的8個區(qū)域；通過使用SYTO 9作為熒光染料實現(xiàn)了對擴增反應(yīng)的實時監(jiān)測,考察了該體系的特異性和檢測靈敏度,并與傳統(tǒng)實時熒光PCR的檢測效果進行了對比,結(jié)果表明：所建立的CPA體系能夠特異性檢測轉(zhuǎn)基因農(nóng)作物中的nos基因；其檢測限約為103拷貝水稻基因組DNA,相比傳統(tǒng)實時熒光PCR檢測,能夠達到相同的檢測限但需要更短的時間(節(jié)約10 min左右)；除此之外,能夠檢測至少0.5%摻雜的實際樣本,足以滿足國際上大部分國家和地區(qū)對轉(zhuǎn)基因檢測的需求。綜上所述,CPA體系能夠作為快速、簡便的核酸擴增方法取代傳統(tǒng)的實時熒光PCR,表現(xiàn)為需要更短的時間、更簡單的反應(yīng)儀器。2)以核酸擴增過程中的副產(chǎn)物H+為檢測對象,建立了基于pH變化檢測的電化學無標記核酸擴增檢測方法,搭建了實時分析和終點檢測兩種形式的檢測平臺,結(jié)果表明：最適合用于pH檢測的CPA體系(pH-CPA)的組成為50mMK+,15mM NH4+,3 mM的KOH添加；使用商業(yè)化pH計可以對pH-CPA擴增進行實時監(jiān)測,將擴增過程中的pH變化最快的點作為pH-CPA反應(yīng)的信號閾值,通過對擴增過程中得到pH變化曲線進行求導,得到曲線的局部最小值即為pH-CPA的信號閾值,信號閾值所對應(yīng)的時間即為"Time to threshold",用tth表示；利用tth分析,pH-CPA擴增方法表現(xiàn)出可靠的定量分析性能,10倍差異的模板,tth大約差3；相比實時熒光檢測的方法,該電化學檢測方法節(jié)省大約20%的時間；此外,通過使用可拋棄的pH敏感的絲網(wǎng)印刷微電極,初始模板量為103拷貝基因組DNA的陽性樣本擴增前后峰值電位差△Ep發(fā)生28.3 mV的變化,對應(yīng)為0.38個pH,與使用商業(yè)化pH計實時分析得到的結(jié)果大致相同(約0.35 pH)。綜上可知,以溶液中的H+濃度的變化為檢測對象能夠?qū)崿F(xiàn)對核酸擴增反應(yīng)實時或是終點的電化學檢測,無需龐大的儀器設(shè)備,與恒溫擴增技術(shù)結(jié)合應(yīng)用,使得核酸分析過程更便攜、簡單易用,由于電化學檢測本身具有易于集成化的特性使得該方法具有更好的應(yīng)用前景。3)通過檢測核酸擴增過程中的Pi信號的產(chǎn)生,建立了基于Pi分析的可視化核酸恒溫擴增檢測方法,詳細系統(tǒng)地考察了該方法應(yīng)用于不同擴增體系的可行性,并設(shè)計了配套的可拋棄的防污染裝置,建立了完整的快速、便攜、可視化核酸擴增檢測體系,結(jié)果表明：核酸擴增條件下非擴增特異性Pi產(chǎn)生的唯一來源是dNTP分解,影響dNTP分解最主要的因素是反應(yīng)溫度條件,傳統(tǒng)PCR(加熱／冷卻熱循環(huán),40 cycles)條件下約13%的dNTP分解產(chǎn)生Pi,而恒溫CPA擴增條件下(63℃,1 h)則僅有2%的dNTP發(fā)生分解；在擴增特異性Pi信號產(chǎn)生情況的研究中,經(jīng)過PPase處理的CPA陽性樣本約產(chǎn)生2.86 mM的Pi,是不經(jīng)過PPase處理的陽性樣本產(chǎn)生量的36倍,陰性樣本產(chǎn)生量的53倍(經(jīng)過PPase處理),而PCR體系陽性樣本(經(jīng)過PPase處理)則產(chǎn)生約0.5 mM的Pi,相應(yīng)陰性樣本(經(jīng)過PPase處理)產(chǎn)生Pi0.15 mM；通過對dNTP利用率的計算可以得到CPA擴增體系中有大約87.5%的‘dNTPs被用于擴增,將近3.36%發(fā)生非擴增特異性降解；而PCR擴增體系則僅有22.3%的dNTPs被用于擴增反應(yīng),18.6%的dNTPs降解;使用該可視化方法檢測CPA,僅需擴增15 min即可完成對103拷貝基因組DNA的檢測,相比第3章基于pH變化的電化學檢測核酸擴增的方法節(jié)約將近50%的時間；通過考察該方法在LAMP擴增中的應(yīng)用,得知該方法并不局限于對CPA擴增體系的檢測,具有能夠應(yīng)用于其他種類恒溫擴增體系的巨大潛力。綜上可知,以溶液中的Pi信號的產(chǎn)生為檢測對象能夠非常直接、簡便地對核酸擴增結(jié)果進行判斷,可視化信號非常明顯、反應(yīng)速度快,更大程度地發(fā)揮了恒溫擴增用于現(xiàn)場、資源匱乏條件下進行核酸分析的優(yōu)勢。4)建立了基于邏輯判斷的轉(zhuǎn)基因品系鑒定方法,以國內(nèi)3種常見抗蟲轉(zhuǎn)基因水稻(華恢1號、科豐6號和克螟稻1號)為研究對象,篩選出能夠有效獲取該組對象基因轉(zhuǎn)化詳細信息的基因組合,以LAMP為恒溫擴增方法示例,通過Mo-Sb-Vc體系對結(jié)果顯色,陽性結(jié)果表現(xiàn)為藍色計作“1”,陰性結(jié)果顯示為無色計作“0”,根據(jù)樣品檢測結(jié)果得到的類似“1101”的結(jié)果進行邏輯判斷,最終鑒定出轉(zhuǎn)基因具體品系,結(jié)果表明,同步檢測sps、nos、 CaMV35S和crylAc基因需要擴增約35min,轉(zhuǎn)基因摻雜度低至0.5%的樣品仍能產(chǎn)生明顯的顏色變化,檢測結(jié)果基本不受深加工工藝過程的影響；使用Whatman 1級Chr層析紙作為基質(zhì),使用磷酸顯色試劑對其進行預(yù)處理,根據(jù)陣列中顯色得到的藍色／無色斑點的數(shù)量及順序即可鑒定出轉(zhuǎn)基因樣本的具體品系。綜上所述,我們建立了一種新型的多基因分析策略用于抗蟲轉(zhuǎn)基因品系鑒定,具有信息量豐富、快速、簡單易用、易于更新的特點,為多目標分析提供新思路。
[Abstract]:In order to develop a simple , rapid and portable method for the detection of transgenic lines , six primers were designed , including two external primers , two cross primers and two detection primers , which specifically identified 8 regions of the ' T - nos sequence .
By using SYTO 9 as the fluorescent dye , real - time monitoring of the amplification reaction was realized , the specificity and the detection sensitivity of the system were investigated , and compared with the traditional real - time fluorescence PCR detection results , the results showed that the established CPA system can specifically detect the nos gene in the transgenic crops ;
the detection limit is about 103 copies of rice genome DNA , compared with the traditional real - time fluorescence PCR detection , the detection limit can reach the same detection limit but shorter time ( about 10 minutes is saved ) ;
In conclusion , the CPA system can be used as a rapid and simple nucleic acid amplification method to replace the traditional real - time fluorescence PCR . In conclusion , the CPA system can be used as a rapid and simple nucleic acid amplification method to replace the traditional real - time fluorescence PCR .
A commercial pH meter can be used for real - time monitoring of pH - CPA amplification , and the pH value in the amplification process is taken as a signal threshold of the pH - CPA reaction , and a pH change curve is obtained through the amplification process to obtain a local minimum value of the curve , namely , the signal threshold value of the pH - CPA , the time corresponding to the signal threshold is " Time to threshold " , and is expressed by tth ;
Using tth analysis , the pH - CPA amplification method showed reliable quantitative analysis performance , 10 - fold difference template , tth about 3 ;
Compared with the real - time fluorescence detection method , the electrochemical detection method saves about 20 percent of time ;
In addition , by using a disposable pH - sensitive screen printing microelectrode , the peak potential difference between the pre - and post - amplification peak potential difference of 103 copies of genomic DNA , which corresponds to 0.38 pH , was approximately the same as the results obtained in real - time analysis using commercial pH meter ( about 0.35 pH ) . It is known that the detection target can realize the electrochemical detection of real - time or end - point of nucleic acid amplification reaction by the change of concentration of H + in solution . The method is more portable and easy to use because electrochemical detection itself has the characteristics of easy integration . The results show that the unique source of non - amplification specific Pi in nucleic acid amplification is the reaction temperature condition .
In the study of the generation of the amplified specific Pi signal , the CPA positive sample treated by PPase was about 2.86 mM Pi , 36 times the amount of positive sample that was not treated by PPase , 53 times the amount of negative sample ( treated by PPase ) , whereas the PCR system - positive sample ( treated with PPase ) produced about 0.5 mM Pi , and the corresponding negative sample ( treated with PPase ) produced Pi0 . 15 mM ;
The results show that the method is not limited to the detection of nucleic acid amplification system . The results show that the method is not limited to the detection of nucleic acid amplification system . The results show that the method is not limited to the detection of nucleic acid amplification system .
【學位授予單位】：浙江大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：S188
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本文編號：2086305