本文選題：轉基因品系 + 阪崎腸桿菌　； 參考：《青島大學》2017年碩士論文

【摘要】：隨著轉基因的安全性成為社會關注的熱點,各國都相繼出臺了關于標識轉基因產品的法規(guī),而市場準入的關鍵就在于定性或者定量的轉基因檢測技術。本研究對轉基因DAS-44406-6大豆品系、轉基因AV43-6-G7馬鈴薯品系分別進行5′-RACE,測出兩個品系外源基因片段和內源基因重組的邊界序列,并按照邊界特異性序列設計出相應的引物探針對,首次建立了轉基因DAS-44406-6大豆品系和轉基因AV43-6-G7馬鈴薯品系的實時熒光PCR和數(shù)字PCR檢測方法。轉基因DAS-44406-6大豆品系和轉基因AV43-6-G7馬鈴薯品系的熒光PCR檢測方法在模板DNA濃度為100 ng/反應時,檢測低限均為0.01%的轉基因含量;建立的轉基因DAS-44406-6大豆品系和轉基因AV43-6-G7馬鈴薯品系數(shù)字PCR檢測方法絕對靈敏度能準確定量到模板DNA濃度分別為0.01 ng/μL和0.001 ng/μL的轉基因樣品。數(shù)字PCR方法相比于傳統(tǒng)PCR檢測方法具有簡便快速、靈敏準確、可絕對定量等特點,這對于轉基因品系的實際檢測具有很大的應用價值。阪崎腸桿菌(Enterobacter sakazakii)可導致免疫力低下的新生兒神經系統(tǒng)紊亂如腦膜炎以及患菌血癥、壞死性小腸結腸炎等疾病,嬰幼兒配方奶粉是至今已知的最主要的感染途徑。而檢測阪崎腸桿菌的關鍵就在于實時熒光PCR和數(shù)字PCR檢測方法。本研究首次建立了微滴式數(shù)字PCR檢測阪崎腸桿菌的絕對定量檢測方法。根據(jù)阪崎腸桿菌的特異性基因序列,設計引物和熒光探針,并進行篩選,和其它12種近緣的、食品中常見的其它菌種一起進行實時熒光PCR引物和探針的特異性以及檢測低限實驗,作為數(shù)字PCR的準備與參照。以阪崎腸桿菌基因組DNA制備絕對靈敏度與相對靈敏度的梯度稀釋DNA進行PCR的定量檢測,并確定和驗證檢測體系的穩(wěn)定性、精密度、線性范圍及檢測低限等指標。本研究建立的檢測阪崎腸桿菌的QX 200數(shù)字PCR檢測方法定量檢測低限為5×10-5 ng/μL,達到實時熒光PCR方法檢測低限,同時在阪崎腸桿菌基因組濃度等于5×10-6 ng/μL時,仍可以檢出病原菌,說明QX 200數(shù)字PCR檢測方法靈敏度達到甚至超過實時熒光PCR方法。樣品DNA模板濃度為5×10-3 ng/μL時,在阪崎腸桿菌基因組DNA相對含量區(qū)間為1.5625%-100%情況下具有較好的定量檢出阪崎腸桿菌的能力;在樣品模板濃度為5×10-3 ng/μL且阪崎腸桿菌在0.78125%-1.5625%時,也可以實現(xiàn)痕量阪崎腸桿菌樣品的檢出。首次建立的數(shù)字PCR檢測方法具有較高的準確性和重復性、較短的檢測時間,對快速檢測出樣品中的阪崎腸桿菌具有重要意義。
[Abstract]:With the safety of GM has become the focus of attention, many countries have issued laws and regulations on the identification of GMOs, and the key to market access is qualitative or quantitative transgenic detection technology. In this study, transgenic DAS-44406-6 soybean lines and transgenic AV43-6-G7 potato lines were carried out 5GRACE-RACE-RACE respectively, and the boundary sequence of exogenous gene fragment and endogenous gene recombination was detected, and the corresponding primer probe pairs were designed according to the boundary specific sequence. Real-time fluorescence PCR and digital PCR detection methods for transgenic DAS-44406-6 soybean and transgenic AV43-6-G7 potato strains were established for the first time. The fluorescence PCR detection method for transgenic DAS-44406-6 soybean lines and transgenic AV43-6-G7 potato lines was 0.01% when the template DNA concentration was 100 ng/. The absolute sensitivity of the established digital PCR detection method for transgenic DAS-44406-6 soybean strains and transgenic AV43-6-G7 potato strains can be accurately quantified to the transgenic samples with template DNA concentrations of 0.01 ng/ 渭 L and 0.001 ng/ 渭 L, respectively. Compared with the traditional PCR detection method, the digital PCR method has the advantages of simple, rapid, sensitive and accurate, and can be used in absolute quantitative analysis, which has great application value for the practical detection of transgenic lines. Enterobacter sakazakii) can lead to neurological disorders such as meningitis, bacteremia, necrotizing enterocolitis and so on. Infant formula is the most important way of infection. The key to detect Enterobacter sakazakii is real-time fluorescent PCR and digital PCR. In this study, an absolute quantitative method for detection of Enterobacter sakazakii by microdrop digital PCR was established for the first time. According to the specific gene sequence of Enterobacter sakazakii, primers and fluorescent probes were designed and screened. The specificity of real-time fluorescent PCR primers and probes and the detection of low limit test were carried out together with other common strains of food as preparation and reference for digital PCR. The absolute sensitivity and relative sensitivity gradient dilution DNA of Enterobacter sakazakii genomic DNA were used to quantitatively detect PCR, and the stability, precision, linear range and detection limit of the detection system were determined and verified. The quantitative detection limit of the QX200 digital PCR method for detection of Enterobacter sakazakii was 5 脳 10 ~ (-5) ng/ 渭 L, which reached the low detection limit by real-time fluorescence PCR method. At the same time, when the genomic concentration of Enterobacter sakazakii was equal to 5 脳 10 ~ (-6) ng/ 渭 L, the pathogen could still be detected. It shows that the sensitivity of QX200 digital PCR detection method is even higher than that of real time fluorescence PCR method. When the concentration of sample DNA template was 5 脳 10-3 ng/ 渭 L, the relative content of genomic DNA of Enterobacter sakazakii was 1.5625- 100%, and when the concentration of template was 5 脳 10-3 ng/ 渭 L and the concentration of Enterobacter sakazakii was 0.78125 and 1.5625%, Trace Enterobacter sakazakii samples can also be detected. The first digital PCR detection method has high accuracy, reproducibility and short detection time, which is of great significance for rapid detection of Enterobacter sakazakii in samples.
【學位授予單位】：青島大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S565.1;S532;R378

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