基于輔助探針與免酶的擴(kuò)增技術(shù)對(duì)病毒DNA的檢測(cè)
發(fā)布時(shí)間：2018-10-12 19:12
【摘要】：HIV病毒和HBV DNA病毒是當(dāng)下研究的熱點(diǎn)。因其對(duì)人類造成摧毀性的害處而聞名于世。DNA是儲(chǔ)存、復(fù)制和傳遞遺傳信息的主要物質(zhì)。檢測(cè)病毒的DNA對(duì)病毒的感染判定、病程監(jiān)控,以及探究疾病內(nèi)源有至關(guān)重要的作用。因此,建立一種快速,有效,簡(jiǎn)便的檢測(cè)病毒DNA的方法非常重要。熒光法以其快速,簡(jiǎn)便,高效等優(yōu)點(diǎn)被廣泛應(yīng)用于核酸檢測(cè)。信號(hào)放大技術(shù)是非常實(shí)用的技術(shù),能夠使檢測(cè)變得更加靈敏。同時(shí)無(wú)酶無(wú)標(biāo)記法能夠減低實(shí)驗(yàn)成本,是實(shí)驗(yàn)變得更加快速。本文還采用了輔助DNA探針進(jìn)行鏈替換反應(yīng),省去了設(shè)計(jì)多條分子信標(biāo)的麻煩。使的本實(shí)驗(yàn)更加簡(jiǎn)便,有效。1、設(shè)計(jì)了一個(gè)發(fā)卡型分子信標(biāo)MB和兩條輔助探針。借助G-四聯(lián)體為信號(hào)顯示平臺(tái)。當(dāng)目標(biāo)物DNA加入時(shí),MB與目標(biāo)物進(jìn)行雜交反應(yīng)。隨后輔助探針與他們的雜交產(chǎn)物進(jìn)行鏈取代反應(yīng),被取代下來(lái)的DNA繼續(xù)和其他MB進(jìn)行反應(yīng),進(jìn)行循環(huán)擴(kuò)增。選用THT染料(硫代黃色素)與G-四聯(lián)體結(jié)合,我們通過(guò)熒光分光光度計(jì)能檢測(cè)到信號(hào)增強(qiáng)了3倍。在目標(biāo)物濃度范圍為(0.1 nM-50 nM)時(shí),取得一個(gè)良好的線性效果。檢出限位50 pM。2、設(shè)計(jì)修飾了二氨基嘌呤的發(fā)卡結(jié)構(gòu)分子信標(biāo)MB.利用二氨基嘌呤在單鏈DNA上顯示熒光,雙鏈上不顯示熒光的特點(diǎn)。加入目標(biāo)物與分子信標(biāo)進(jìn)行雜交鏈反應(yīng),熒光法檢測(cè)到2氨基嘌呤釋放熒光。當(dāng)用輔助探針將目標(biāo)物替換下來(lái)后,目標(biāo)物繼續(xù)與分子信標(biāo)MB反應(yīng),產(chǎn)生大量的能夠釋放二氨基嘌呤產(chǎn)物。信號(hào)擴(kuò)增了3.4倍。無(wú)需熒光猝滅。檢出限為80 pM。3、設(shè)計(jì)了末端能夠形成G-四聯(lián)體結(jié)構(gòu)的分子信標(biāo)H1以及另外一條分子信標(biāo)H2。NMM(N-甲基卟啉二丙酸IX)與其結(jié)合產(chǎn)生一定熒光信號(hào)。目標(biāo)物與分子信標(biāo)H1的結(jié)合使分子信標(biāo)打開(kāi),G-四聯(lián)體結(jié)構(gòu)不能形成。熒光下降。H2與其雜交產(chǎn)物進(jìn)行鏈取代反應(yīng),以致目標(biāo)物循環(huán)。熒光下降4倍。檢出限為65 pM。
[Abstract]:HIV virus and HBV DNA virus are the focus of current research. Known for its devastating effects on humans, DNA is the main material for storing, copying, and transmitting genetic information. Detection of virus DNA plays an important role in virus infection detection, disease course monitoring, and disease endogenous detection. Therefore, it is very important to establish a rapid, effective and simple method for the detection of viral DNA. Fluorescence method has been widely used in nucleic acid detection for its advantages of fast, simple and high efficiency. Signal amplification is a very practical technique, which can make detection more sensitive. At the same time, enzyme-free labeling can reduce the cost of the experiment, so the experiment becomes faster. An auxiliary DNA probe is also used for chain substitution reaction, which saves the trouble of designing multiple molecular beacons. This experiment is more simple and effective. 1. A card type molecular beacon MB and two auxiliary probes are designed. G-quadruple is used as signal display platform. When the target DNA was added, the MB was hybridized with the target. Then the auxiliary probes reacted with their hybridization products and the replaced DNA continued to react with other MB for cyclic amplification. By using THT dye (thioyellow pigment) combined with G-quadruple, we can detect the signal by three times by fluorescence spectrophotometer. A good linear effect was obtained when the concentration of the target was (0. 1 nM-50 nM). The detection limit of 50 pM.2, was designed to modify the molecular beacon MB. of diaminopurine. Using diaminopurine to display fluorescence on single strand DNA, no fluorescence on double strand. The 2 aminopurine was detected by fluorescence method in the hybridization chain reaction between the target and the molecular beacon. When the target was replaced by an auxiliary probe, the target continued to react with the molecular beacon MB to produce a large amount of diaminopurine products. The signal amplification was 3.4 times. There is no need for fluorescence quenching. The detection limit of 80 pM.3, was used to design a molecular beacon H1 which could form a G-quadruplex structure and another molecular beacon H2.NMM (N-methylporphyrin dipropionate IX) to produce a certain fluorescence signal. The binding of the target and the molecular beacon H 1 makes the molecular beacon open and the G- quadruple structure cannot be formed. The fluorescence of H _ 2 was decreased. The H _ 2 chain substitution reaction was carried out with its hybrid product, which resulted in the circulation of the target material. The fluorescence decreased by four times. Detection limit is 65 pM.
【學(xué)位授予單位】：湘潭大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R446.6
本文編號(hào)：2267287
[Abstract]:HIV virus and HBV DNA virus are the focus of current research. Known for its devastating effects on humans, DNA is the main material for storing, copying, and transmitting genetic information. Detection of virus DNA plays an important role in virus infection detection, disease course monitoring, and disease endogenous detection. Therefore, it is very important to establish a rapid, effective and simple method for the detection of viral DNA. Fluorescence method has been widely used in nucleic acid detection for its advantages of fast, simple and high efficiency. Signal amplification is a very practical technique, which can make detection more sensitive. At the same time, enzyme-free labeling can reduce the cost of the experiment, so the experiment becomes faster. An auxiliary DNA probe is also used for chain substitution reaction, which saves the trouble of designing multiple molecular beacons. This experiment is more simple and effective. 1. A card type molecular beacon MB and two auxiliary probes are designed. G-quadruple is used as signal display platform. When the target DNA was added, the MB was hybridized with the target. Then the auxiliary probes reacted with their hybridization products and the replaced DNA continued to react with other MB for cyclic amplification. By using THT dye (thioyellow pigment) combined with G-quadruple, we can detect the signal by three times by fluorescence spectrophotometer. A good linear effect was obtained when the concentration of the target was (0. 1 nM-50 nM). The detection limit of 50 pM.2, was designed to modify the molecular beacon MB. of diaminopurine. Using diaminopurine to display fluorescence on single strand DNA, no fluorescence on double strand. The 2 aminopurine was detected by fluorescence method in the hybridization chain reaction between the target and the molecular beacon. When the target was replaced by an auxiliary probe, the target continued to react with the molecular beacon MB to produce a large amount of diaminopurine products. The signal amplification was 3.4 times. There is no need for fluorescence quenching. The detection limit of 80 pM.3, was used to design a molecular beacon H1 which could form a G-quadruplex structure and another molecular beacon H2.NMM (N-methylporphyrin dipropionate IX) to produce a certain fluorescence signal. The binding of the target and the molecular beacon H 1 makes the molecular beacon open and the G- quadruple structure cannot be formed. The fluorescence of H _ 2 was decreased. The H _ 2 chain substitution reaction was carried out with its hybrid product, which resulted in the circulation of the target material. The fluorescence decreased by four times. Detection limit is 65 pM.
【學(xué)位授予單位】：湘潭大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R446.6
【參考文獻(xiàn)】
相關(guān)期刊論文 前2條
1 Giuseppe La Torre;Rosella Saulle;;Risk factors from HBV infection among blood donors:A systematic review[J];Asian Pacific Journal of Tropical Biomedicine;2016年04期
2 Moon S Chen Jr;Julie Dang;;Hepatitis B among Asian Americans:Prevalence, progress, and prospects for control[J];World Journal of Gastroenterology;2015年42期
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