一种新型核酸恒温扩增方法的研究及其在现场检测中的应用

发布时间：2018-06-30 08:00

本文选题：核酸恒温扩增技术 + 现场快速检测　；参考：《中国人民解放军军事医学科学院》2016年博士论文

【摘要】：近些年来,新发、突发传染病疫情不断增多,传统传染病有不断抬头的趋势,对人民生命财产安全构成了极大的危害,与此同时我国地震、洪涝灾害频发,且随着中国国际化进程的加快,在华举办的各种国际重大会议及活动增多,安保任务加重,在这样的背景下,对病原微生物的现场检测(point-of-care testing,POCT)提出了很高的要求。病原微生物的现场检测技术主要包括免疫学检测和核酸检测两大类,由于免疫学方法总体灵敏度不高,故又以核酸检测为主。核酸检测按反应温度的不同分为变温核酸扩增技术(Alternating Temperature Nucleic Acid Testing,ATNAT)和恒温核酸扩增技术(Isothermal Nucleic Acid Testing,INAT)。ATNAT主要是聚合酶链式反应(Polymerase Chain Reaction,PCR)及其衍生技术,如荧光定量PCR、逆转录PCR等,这些技术共同特点是需要温控严格、元件精密的复杂仪器。INAT是指在恒定温度下扩增核酸的技术,由于温度要求单一,INAT反应在恒温仪器中就可以进行,如水浴锅、金属浴甚至一个保温效果好的保温杯就可以完成反应,彻底抛弃了PCR等技术需要的变温装置,简化了操作步骤,满足部队野战、社区基层医疗、现场流行病学调查、医院床头诊断等即时检测环境下的需求。常用的INAT技术主要包括链替代扩增(SDA)、滚环扩增法(RCA)、环介导恒温核酸扩增法(LAMP)、重组酶聚合酶扩增(RPA)等技术,其中LAMP法由于只需一种酶、扩增效率高,是目前INAT技术中应用最多、论文发表量最大的方法,但也有着引物设计复杂、假阳性率偏高、试剂价格高等缺点,且由于日本知识产权的保护,我国在转化应用上局限性较强。为此我们发明了聚合酶螺旋反应(Polymerase Spiral Reaction,PSR)这一新型的核酸恒温扩增方法,它利用混合引物的设计思路和Bst DNA聚合酶的链置换活性,通过引物结合、延伸、解链、单链旋转、再次延伸的循环,达到等温条件下核酸扩增的目的,PSR兼具PCR法引物设计简单和LAMP法只需一种酶、反应高效的特点,首次实现了一对引物和一种酶在等温环境下的核酸扩增。为进一步提高PSR法的反应速率,我们引入了加速引物的概念,使得反应Ct值缩小到20min之内,并优化了反应体系和引物设计参数,最终建立聚合酶螺旋反应这一新型的核酸恒温扩增方法。常用的核酸检测结果判读方法包括琼脂糖凝胶电泳法、荧光成像法、核酸薄膜层析试纸条法和实时浊度法等方法,但它们都有一个共同的特点即需要专业的仪器,且对操作人员专业水平要求较高,在现场检测中局限性较强。本研究利用PSR反应中金属离子浓度、酸碱性等反应体系参数的变化,采用钙黄绿素(Calcein)、羟基萘酚蓝(HNB)和pH值指示剂(甲酚红-苯酚红)这三种颜色指示剂,评估其对反应的影响及最佳显色浓度。最终在反应前加入1μl指示剂,反应后通过简单的颜色判读即可判断反应结果,阴性到阳性的变化分别为钙黄绿素(淡橙色→绿色)、羟基萘酚蓝(紫色→天蓝色)、甲酚红-苯酚红(红色→黄色),PSR显色法简便、经济、肉眼可识别,完全满足POCT检测的要求。从待测生物样本中提取核酸是核酸检测的第一步,现场、临床等即时检测环境下也不例外。核酸提取一般是采用基于胍盐裂解法、硅膜法、磁珠法等各种原理的试剂盒,或者是采用核酸自动化提取仪,但用试剂盒提取核酸费时费力、操作复杂,往往需要2小时以上的时间,不能满足即时检测快速、简便的需求,核酸自动化提取仪能实现高通量且操作简便,但比较笨重、维护成本高。本研究通过核酸暴露的理论,研制了一种以非离子型表面活性剂Chelex-100、TritonX-100为主要裂解成分的样本处理管,评价其从革兰氏阳性菌、革兰氏阴性菌、真菌、病毒等不同病原微生物中提取核酸的效果,结果显示只需要在90℃裂解10min,即可适用于PSR、LAMP及荧光定量PCR等检测技术,并且效果与商用试剂盒相当,具有操作简便、裂解效果好、不影响后续反应的特点,且不受粪便、痰液、血液等复杂样本的影响,同时具有病原灭活、核酸释放、过滤除杂、精确加样这四大特点,特别适合于现场检测情况下使用。酶、引物等生物活性物质在非低温环境下易失活,通过与国内同行的交流与合作,采用玻璃化技术对PSR反应体系中的生物活性物质进行处理,实验结果证实处理后的酶在50℃放置加速破坏,2个月后活性没有明显变化,相当于室温放置1-2年,解决了这一问题。并研制了便携式PSR检测仪,检测仪同时包括裂解区和扩增区,通过挤压样本处理管滴加一滴裂解液(约10μl)至反应管盖上,静置片刻后甩下液体在检测仪中完成扩增,在提示声响后取出即可通过颜色变化判读阴阳性结果,便携式PSR检测仪通过了不同酸碱度样本和粪便、尿液、血液等复杂样本的考核。本研究在完善PSR反应及其相应的样本快速处理、结果快速判读等方法后,建立了基于PSR反应的快速检测技术平台,为日常疾病预防控制任务和申请国家药监局相应批号提供技术支持,并致力于应对新发、突发传染病疫情及实验室常规的病原检测。本研究还构建了肺炎克雷伯菌、白色念珠菌、铜绿假单胞菌、肠道病毒71型、柯萨奇病毒A16型、霍乱弧菌这六种病原微生物的PSR检测方法,实验数据证实其具有良好的特异性和敏感性,并在临床考核中取得了良好成效。综上所述,本研究发明了聚合酶螺旋反应这一新型的核酸恒温扩增方法,具有良好的特异性与敏感性;解决了样本核酸提取和扩增结果判读繁琐、复杂的问题;建立了基于PSR反应的快速检测技术平台;并研制了简洁易用的便携式PSR检测仪。我们认为PSR技术将为即时检测、应对新发突发传染病疫情及实验室常规的病原检测提供有力的技术支持,逐渐走向现场、走向基层、走向家庭,最终让核酸检测随处可行。
[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.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R440

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