發(fā)布時間：2018-06-30 08:00

  本文選題：核酸恒溫擴增技術(shù) + 現(xiàn)場快速檢測　； 參考：《中國人民解放軍軍事醫(yī)學(xué)科學(xué)院》2016年博士論文

【摘要】：近些年來,新發(fā)、突發(fā)傳染病疫情不斷增多,傳統(tǒng)傳染病有不斷抬頭的趨勢,對人民生命財產(chǎn)安全構(gòu)成了極大的危害,與此同時我國地震、洪澇災(zāi)害頻發(fā),且隨著中國國際化進程的加快,在華舉辦的各種國際重大會議及活動增多,安保任務(wù)加重,在這樣的背景下,對病原微生物的現(xiàn)場檢測(point-of-care testing,POCT)提出了很高的要求。病原微生物的現(xiàn)場檢測技術(shù)主要包括免疫學(xué)檢測和核酸檢測兩大類,由于免疫學(xué)方法總體靈敏度不高,故又以核酸檢測為主。核酸檢測按反應(yīng)溫度的不同分為變溫核酸擴增技術(shù)(Alternating Temperature Nucleic Acid Testing,ATNAT)和恒溫核酸擴增技術(shù)(Isothermal Nucleic Acid Testing,INAT)。ATNAT主要是聚合酶鏈式反應(yīng)(Polymerase Chain Reaction,PCR)及其衍生技術(shù),如熒光定量PCR、逆轉(zhuǎn)錄PCR等,這些技術(shù)共同特點是需要溫控嚴格、元件精密的復(fù)雜儀器。INAT是指在恒定溫度下擴增核酸的技術(shù),由于溫度要求單一,INAT反應(yīng)在恒溫儀器中就可以進行,如水浴鍋、金屬浴甚至一個保溫效果好的保溫杯就可以完成反應(yīng),徹底拋棄了PCR等技術(shù)需要的變溫裝置,簡化了操作步驟,滿足部隊野戰(zhàn)、社區(qū)基層醫(yī)療、現(xiàn)場流行病學(xué)調(diào)查、醫(yī)院床頭診斷等即時檢測環(huán)境下的需求。常用的INAT技術(shù)主要包括鏈替代擴增(SDA)、滾環(huán)擴增法(RCA)、環(huán)介導(dǎo)恒溫核酸擴增法(LAMP)、重組酶聚合酶擴增(RPA)等技術(shù),其中LAMP法由于只需一種酶、擴增效率高,是目前INAT技術(shù)中應(yīng)用最多、論文發(fā)表量最大的方法,但也有著引物設(shè)計復(fù)雜、假陽性率偏高、試劑價格高等缺點,且由于日本知識產(chǎn)權(quán)的保護,我國在轉(zhuǎn)化應(yīng)用上局限性較強。為此我們發(fā)明了聚合酶螺旋反應(yīng)(Polymerase Spiral Reaction,PSR)這一新型的核酸恒溫擴增方法,它利用混合引物的設(shè)計思路和Bst DNA聚合酶的鏈置換活性,通過引物結(jié)合、延伸、解鏈、單鏈旋轉(zhuǎn)、再次延伸的循環(huán),達到等溫條件下核酸擴增的目的,PSR兼具PCR法引物設(shè)計簡單和LAMP法只需一種酶、反應(yīng)高效的特點,首次實現(xiàn)了一對引物和一種酶在等溫環(huán)境下的核酸擴增。為進一步提高PSR法的反應(yīng)速率,我們引入了加速引物的概念,使得反應(yīng)Ct值縮小到20min之內(nèi),并優(yōu)化了反應(yīng)體系和引物設(shè)計參數(shù),最終建立聚合酶螺旋反應(yīng)這一新型的核酸恒溫擴增方法。常用的核酸檢測結(jié)果判讀方法包括瓊脂糖凝膠電泳法、熒光成像法、核酸薄膜層析試紙條法和實時濁度法等方法,但它們都有一個共同的特點即需要專業(yè)的儀器,且對操作人員專業(yè)水平要求較高,在現(xiàn)場檢測中局限性較強。本研究利用PSR反應(yīng)中金屬離子濃度、酸堿性等反應(yīng)體系參數(shù)的變化,采用鈣黃綠素(Calcein)、羥基萘酚藍(HNB)和pH值指示劑(甲酚紅-苯酚紅)這三種顏色指示劑,評估其對反應(yīng)的影響及最佳顯色濃度。最終在反應(yīng)前加入1μl指示劑,反應(yīng)后通過簡單的顏色判讀即可判斷反應(yīng)結(jié)果,陰性到陽性的變化分別為鈣黃綠素(淡橙色→綠色)、羥基萘酚藍(紫色→天藍色)、甲酚紅-苯酚紅(紅色→黃色),PSR顯色法簡便、經(jīng)濟、肉眼可識別,完全滿足POCT檢測的要求。從待測生物樣本中提取核酸是核酸檢測的第一步,現(xiàn)場、臨床等即時檢測環(huán)境下也不例外。核酸提取一般是采用基于胍鹽裂解法、硅膜法、磁珠法等各種原理的試劑盒,或者是采用核酸自動化提取儀,但用試劑盒提取核酸費時費力、操作復(fù)雜,往往需要2小時以上的時間,不能滿足即時檢測快速、簡便的需求,核酸自動化提取儀能實現(xiàn)高通量且操作簡便,但比較笨重、維護成本高。本研究通過核酸暴露的理論,研制了一種以非離子型表面活性劑Chelex-100、TritonX-100為主要裂解成分的樣本處理管,評價其從革蘭氏陽性菌、革蘭氏陰性菌、真菌、病毒等不同病原微生物中提取核酸的效果,結(jié)果顯示只需要在90℃裂解10min,即可適用于PSR、LAMP及熒光定量PCR等檢測技術(shù),并且效果與商用試劑盒相當,具有操作簡便、裂解效果好、不影響后續(xù)反應(yīng)的特點,且不受糞便、痰液、血液等復(fù)雜樣本的影響,同時具有病原滅活、核酸釋放、過濾除雜、精確加樣這四大特點,特別適合于現(xiàn)場檢測情況下使用。酶、引物等生物活性物質(zhì)在非低溫環(huán)境下易失活,通過與國內(nèi)同行的交流與合作,采用玻璃化技術(shù)對PSR反應(yīng)體系中的生物活性物質(zhì)進行處理,實驗結(jié)果證實處理后的酶在50℃放置加速破壞,2個月后活性沒有明顯變化,相當于室溫放置1-2年,解決了這一問題。并研制了便攜式PSR檢測儀,檢測儀同時包括裂解區(qū)和擴增區(qū),通過擠壓樣本處理管滴加一滴裂解液(約10μl)至反應(yīng)管蓋上,靜置片刻后甩下液體在檢測儀中完成擴增,在提示聲響后取出即可通過顏色變化判讀陰陽性結(jié)果,便攜式PSR檢測儀通過了不同酸堿度樣本和糞便、尿液、血液等復(fù)雜樣本的考核。本研究在完善PSR反應(yīng)及其相應(yīng)的樣本快速處理、結(jié)果快速判讀等方法后,建立了基于PSR反應(yīng)的快速檢測技術(shù)平臺,為日常疾病預(yù)防控制任務(wù)和申請國家藥監(jiān)局相應(yīng)批號提供技術(shù)支持,并致力于應(yīng)對新發(fā)、突發(fā)傳染病疫情及實驗室常規(guī)的病原檢測。本研究還構(gòu)建了肺炎克雷伯菌、白色念珠菌、銅綠假單胞菌、腸道病毒71型、柯薩奇病毒A16型、霍亂弧菌這六種病原微生物的PSR檢測方法,實驗數(shù)據(jù)證實其具有良好的特異性和敏感性,并在臨床考核中取得了良好成效。綜上所述,本研究發(fā)明了聚合酶螺旋反應(yīng)這一新型的核酸恒溫擴增方法,具有良好的特異性與敏感性;解決了樣本核酸提取和擴增結(jié)果判讀繁瑣、復(fù)雜的問題;建立了基于PSR反應(yīng)的快速檢測技術(shù)平臺;并研制了簡潔易用的便攜式PSR檢測儀。我們認為PSR技術(shù)將為即時檢測、應(yīng)對新發(fā)突發(fā)傳染病疫情及實驗室常規(guī)的病原檢測提供有力的技術(shù)支持,逐漸走向現(xiàn)場、走向基層、走向家庭,最終讓核酸檢測隨處可行。
[Abstract]:In recent years, the outbreak of infectious diseases has been increasing, the trend of the traditional infectious diseases has been increasing, and the safety of people's life and property has been greatly endangered. At the same time, China's earthquake, flood and waterlogging disasters are frequent, and with the acceleration of the process of internationalization of China, the number of major international conferences and activities in China and security tasks in China have increased. In this context, the field detection of pathogenic microbes (point-of-care testing, POCT) is highly required. The field detection techniques of pathogenic microbes mainly include two major categories of immunological detection and nucleic acid detection, because the overall sensitivity of immunological methods is not high, so nucleic acid detection is the main method. The difference is divided into Alternating Temperature Nucleic Acid Testing, ATNAT and constant temperature nucleic acid amplification technology (Isothermal Nucleic Acid Testing, INAT).ATNAT mainly is polymerase chain reaction and its derivatives, such as fluorescent quantitation, reverse transcriptase, etc. The characteristic is that the complex instrument,.INAT, which needs strict temperature control and precise component, refers to the technology of amplifying nucleic acid at constant temperature. Because of the single temperature requirement, the INAT reaction can be carried out in the constant temperature instrument, such as the water bath pot, the metal bath and even a thermal insulation cup which has good thermal insulation effect, and completely discards the change of the technical needs of PCR and so on. The temperature device simplifies the operation steps to meet the needs of the army field warfare, community grassroots medical treatment, field epidemiological investigation, hospital bedside diagnosis and so on. The commonly used INAT techniques include chain substitution amplification (SDA), rolling ring amplification (RCA), ring mediated constant temperature nucleic acid amplification (LAMP), recombinant enzyme polymerase amplification (RPA), and so on. The medium LAMP method, because only one kind of enzyme is needed, has high amplification efficiency, which is the most widely used method in INAT technology at present. But the design of the primer is complex, the false positive rate is high and the price of the reagent is high. And because of the protection of Japanese intellectual property, our country has a strong limitation in the conversion application. Therefore, we invented the polymerase chain screw. Polymerase Spiral Reaction (PSR), a new type of nucleic acid constant temperature amplification method, uses the design idea of mixed primers and the chain replacement activity of Bst DNA polymerase, through the combination of primers, extension, chain removal, single strand rotation, and the repeat extension of the cycle to achieve the aim of nucleic acid amplification under isothermal conditions, and PSR with PCR primers design is simple. In order to further improve the reaction rate of the PSR method, we introduce the concept of accelerated primers to reduce the reaction Ct value to the 20min, and optimize the reaction system and primer design parameters, and finally build up the poly (LAMP) method. The new method of nucleic acid isothermal amplification of synthase helix reaction. The commonly used methods for nucleic acid detection include agarose gel electrophoresis, fluorescence imaging, nucleic acid film chromatography test paper and real time turbidimetry, but they all have a common feature that requires professional instruments and professional requirements for operators. The three color indicators, such as calcium yellow green (Calcein), hydroxyl naphthol blue (HNB) and pH value indicator (cresol red phenol red), are used to evaluate the effect of the three color indicators on the reaction and the optimum color concentration. 1 mu l indicator should be added before the reaction, and the reaction results can be judged by simple color judgment after reaction. The negative to positive changes are calcium yellowish green (light orange to green), hydroxyl naphthol blue (purple to sky blue), cresol red phenol red (red to yellow), PSR colorimetric method is simple, economical, and visible to the naked eye, and fully meet the requirements of POCT detection. The extraction of nucleic acid from the tested biological samples is the first step in the detection of nucleic acid. It is no exception in the immediate detection environment, such as in situ and clinical. The nucleic acid extraction is usually based on various principles, such as guanidine pyrolysis, silicon membrane, magnetic beads, etc., or the use of nucleic acid automation extraction apparatus, but the time consuming and laborious operation of the nucleic acid extraction kit is carried out. It is complicated, which often takes more than 2 hours, and can not meet the rapid and convenient needs of instant detection. The automated extraction instrument of nucleic acid can achieve high throughput and easy operation, but it is heavy and costly. In this study, a non ionized surface active agent Chelex-100, TritonX-100 is the main lysis through the theory of nucleic acid exposure. The sample processing tube is used to evaluate the effect of nucleic acid extraction from gram-positive bacteria, Gram-negative bacteria, fungi, virus and other pathogenic microbes. The results show that it is suitable for PSR, LAMP and fluorescence quantitative PCR detection techniques at 90 degrees centigrade, and the effect is similar to that of commercial kits. It is easy to operate and cleavage. It has good effect, and does not affect the characteristics of subsequent reactions, and is not affected by complex samples such as feces, sputum, blood and so on. It also has four major characteristics, which are pathogenic inactivation, nucleic acid release, filtration and impurity removal. It is especially suitable for use in field detection. Enzyme, primers and other raw materials are easily inactive in the non cryogenic environment, by the same as in the same country. The biological active substances in the PSR reaction system were treated with vitrification technology. The experimental results confirmed that the treated enzyme was placed at 50 C to accelerate the destruction. After 2 months, the activity did not change obviously, which was equivalent to 1-2 years at room temperature. The portable PSR detector was developed, and the detector included the detector at the same time. The lysate area and the amplification area are treated with a drop of a drop of lysate (about 10 L) to the cover of the reaction tube by the extrusion sample, and then the liquid can be amplified in the detector for a moment, and the color changes can be used to judge the negative and positive results. The portable PSR detector has passed the different pH samples and feces, urine, and blood. After improving the PSR response and its corresponding sample rapid processing and rapid interpretation, this study established a rapid detection technology platform based on PSR reaction, providing technical support for daily disease prevention and control tasks and applying for the corresponding batch number of the National Drug Administration Bureau, and was committed to dealing with new hair and sudden infectious diseases. This study also constructed a PSR detection method for Klebsiella pneumoniae, Candida albicans, Pseudomonas aeruginosa, enterovirus 71, Coxsackie virus A16, Vibrio cholerae, and the experimental data confirmed that it has good specificity and sensitivity, and has been obtained in clinical examination. In summary, the new method of polymerase chain reaction, a new method of nucleic acid constant temperature amplification, has been invented, which has good specificity and sensitivity, and solves the complicated and complicated problem of nucleic acid extraction and amplification. The rapid detection technology platform based on PSR reaction is set up, and a simple and easy to use portable platform is developed. PSR detector. We think that PSR technology will be an instant test to provide strong technical support for new outbreak of sudden infectious diseases and laboratory routine pathogen detection, gradually to the scene, to the grass-roots level, to the family, and finally make the nucleic acid detection everywhere feasible.
【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R440

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