一种新的可视化环介导等温扩增技术检测疟原虫的研究及其应用评价

发布时间：2018-01-02 03:00

本文关键词：一种新的可视化环介导等温扩增技术检测疟原虫的研究及其应用评价　出处：《江苏省血吸虫病防治研究所》2012年硕士论文　论文类型：学位论文

【摘要】：疟疾是由疟原虫引起的严重危害人民健康和生命的重大传染病，被列为全球三大公共卫生问题之一。疟疾也曾是严重危害我国人民身体健康和影响社会经济的重要传染病。在上世纪60年代和70年代初，我国中部地区曾两次暴发大范围的疟疾流行，在2000年前后，中部地区再次出现了间日疟疫情回升和局部暴发流行。在长期的不懈努力下，我国的疟疾防治工作取得了巨大成绩，上世纪90年代在中国中部地区已成功消除了恶性疟，到2009年，我国已有效控制了间日疟疫情回升和局部暴发流行，疟疾发病率已降至历史最低水平。我国从2010年开始实施《中国消除疟疾行动计划》。消除疟疾阶段的目标、策略和措施与控制阶段有很大的不同。在疟疾控制阶段，控制的目标是降低疟疾发病率，而在消除疟疾阶段，消除的目标是没有本地感染的疟疾病例，因此，及时和准确发现每一个可能的传染源是阻断疟疾传播的关键。《消除疟疾指导手册》在消除疟疾阶段要求对所有疑似病例进行疟疾实验室检测。但随着疟疾发病率的降低，临床患者血液中原虫密度呈下降趋势，，低原虫密度临床疟疾的比例逐年上升，给疟疾的实验室诊断带来了新的挑战。传统的血涂片镜检是疟疾实验室诊断最常见的方法，但是镜检的敏感性和准确性依赖于镜检人员的技术和经验，且对低原虫密度检测敏感性不高。新发展的疟疾快速诊断技术（RDTs）具有简单和快速的优点，但现有RDTs对低原虫密度感染的检出率，尤其是对间日疟原虫的检测敏感性常较低。巢式PCR等基因检测技术具有高敏感性、高特异性的优点，然而其需要特殊仪器和试剂，不仅价格较昂贵，且操作步骤较复杂，目前尚需要在有条件的中心实验室开展检测。环介导等温扩增技术（LAMP）是一种新的基因检测技术，具有高敏感性、高特异性和操作简便的优势。在具有链置换功能的Bst DNA聚合酶存在下，经过一个恒温扩增的步骤即可完成，能在短时间内实现109-1010的扩增，并产生一种焦磷酸镁衍生物，使反应液呈现肉眼可见的白色浑浊，从而可以根据反应液浑浊与否判定结果。在LAMP反应体系中加入钙黄绿素和核酸染料SYBR Green I可提高观察效果。但钙黄绿素敏感性不足，SYBR Green I对扩增反应有抑制作用，需要在反应后加入，然而由于LAMP的高效性，导致反应后开盖存在扩增产物污染的极大风险。为解决上述问题，本实验室研发的一种针对间日疟原虫的可肉眼观察结果的LAMP检测方法，在扩增前加入含有SYBR Green I染料微晶蜡丸，并在反应完成后可通过加热融化释放染料，使反应产物染色并为肉眼所见。同时蜡丸在试管表面重新凝固形成屏障，可以防止反应产物的污染。但该可视化LAMP技术只能检测间日疟原虫感染，检测的稳定性也有待进一步提高。考虑到目前我国主要存在的除本地感染间日疟病例外，外出务工人员感染输入性恶性疟原虫、间日疟原虫、三日疟原虫和卵形疟原虫的病例也正在逐年增加，且绝大多数的疟疾病例都发生在贫困的偏远地区，迫切需要发展一种能同时检测四种疟原虫，稳定、敏感、特异、操作简便、成本低廉且能够进行大规模检测的新技术以用于疟原虫的检测。本研究在已有的可视化LAMP的基础上进行改进和优化，以应用于对多种疟原虫的检测，同时将改进的可视化LAMP试剂和市售LAMP反应试剂盒进行成本-效果比较以评价其现场推广可能性，并通过对现场样本的检测以评估其现场应用价值。研究包括三个部分：一、可视化环介导等温扩增（LAMP）检测疟原虫技术的改进方法：针对疟原虫18S rRNA基因和线粒体基因属特异性保守区域在线设计引物，和文献报道的引物进行比较和筛选。对反应体系的反应温度、dNTPs浓度、MgSO4浓度、内引物浓度等进行优化。对全血和滤纸血样本分别采用DNA提取试剂盒和简化的加热处理方法提取样本DNA，评价改进的可视化LAMP技术对不同方法处理的样本的扩增效果。以恶性疟原虫、间日疟原虫、三日疟原虫、卵形疟原虫的全血DNA作为模板来评价改进的可视化LAMP检测疟原虫技术的特异性。以质粒梯度浓度和全血样本模拟梯度疟原虫密度样本作为模板，评估改进的可视化LAMP技术的敏感性。结果：针对疟原虫18S rRNA基因和线粒体基因属特异性保守区域在线设计出8对引物，经过比较和筛选，选用文献报道的Pg-18S rRNA引物为最适引物。经优化后改进的可视化LAMP的反应体系和反应条件为：20mM Tris-HCl pH8.8,10mM KCl,10mM (NH4)2SO4,0.1%Tween20、8U Bst DNA聚合酶、1.4mMdNTPs、9mM MgSO4、0.2μM F3和B3、2.5μM FIP和BIP、0.8μM LPF和LPB、1.0μL模板DNA，加DDW至20μL64℃反应60min。改进的可视化LAMP检测疟原虫的方法可特异地检出四种疟原虫；对不同方法处理的样本进行检测均有较好的扩增效果；最低可检测出3.1×100copy/μL的疟原虫18s rRNA基因质粒DNA，扩增反应的Tt值随质粒浓度的递增按照相关关系递减；对模拟的梯度疟原虫密度全血样本最低可检测出4.863p/μL的全血样本。二、可视化LAMP和市售LAMP试剂盒的成本-效果比较方法：分别用自配试剂的可视化LAMP和市售的LAMP试剂盒对疟原虫样本进行检测，对两种LAMP检测方法的反应效率、敏感性和特异性进行检测效果的比较，并对两种检测方法的试剂成本进行比较，评价新的可视化LAMP技术用于检测疟原虫的方法在现场推广使用的可能性。结果：新的可视化LAMP技术和采购的LAMP反应试剂盒用于对疟原虫检测的特异性均较高，但新的可视化LAMP扩增效率更为优异和稳定；优化的可视化LAMP的检测敏感性（4.863p/μL）比LAMP试剂盒检测敏感性48.63p/μL高十倍；自配试剂的可视化LAMP技术检测疟原虫的检测单份样本试剂成本约为7元/份，LAMP反应试剂盒的成本约为166元/份，前者约为后者的1/24。三、可视化环介导等温扩增技术检测疟疾现场样本的应用评价方法：采用新的可视化LAMP技术对现场的滤纸血样本进行检测，并将结果与镜检结果进行比较和分析，对结果不一致的样本分别进行镜检复核、巢式PCR基因复核、样本DNA浓度检测和病例追踪调查复核，评价新的可视化LAMP检测疟原虫技术在我国消除疟疾阶段的现场应用价值。结果：采用新的可视化LAMP技术检测临床采集的网络报告疟疾病例滤纸血样本200份，检测到疟原虫阳性样本186份，阴性样本14份，与镜检结果的符合率为96.5%。与镜检结果不一致的所有样本经巢式PCR复核，结果与LAMP检测结果一致。3份经LAMP检测和巢式PCR复核阳性，但镜检为阴性的样本经省级镜检专家镜检复核后1份为间日疟阳性，但原虫密度很低（16p/μL）；另2份经镜检复核仍为阴性的样本经现场流行病学个案病例追踪调查，确认均为输入性恶性疟病例，并在采血涂片之前均服用过抗疟药物；4份LAMP检测和巢式PCR复核为阴性但镜检为阳性的样本经省级镜检专家镜检复核均为阳性，但经样本DNA浓度检测发现没有提取出DNA。结论 1、本研究选用疟原虫属特异性的18S rRNA引物，建立了一种可用于检测四种疟原虫的可视化LAMP检测技术，检测敏感性达到4.863p/μL。 2、新的可视化LAMP检测技术与市售LAMP反应试剂盒相比，两种方法对疟原虫的检测特异性均较高，但新的可视化LAMP扩增效率和敏感性均显著高于市售LAMP，费用仅为市售LAMP反应试剂盒的1/24。 3、新的可视化LAMP检测技术对现场采集的滤纸血样本的检测结果表明具有比镜检更高的检测敏感性，可用于常规镜检很难检测的低原虫密度和已服用过抗疟药物患者血样的检测。 4、新的可视化LAMP检测技术具有敏感、高效、简便、快捷和可批量检测的优点，可作为疟原虫基因检测的一个新工具，在我国消除疟疾阶段中推广应用。
[Abstract]:Malaria is caused by parasites seriously endanger people's health and life of the major infectious diseases, is listed as one of the world's three major public health problems. Malaria was also a serious harm to the health of our people and the social and economic impact of infectious diseases. In the last century and the beginning of 70s 60s, the malaria epidemic in the middle area of China was two in a large range, before and after 2000, the central region again rebounded vivax malaria epidemic and outbreak. In the long-term unremitting efforts, the work of malaria prevention and control in China has made great achievements, in 90s in the central region of China has successfully eliminated the pernicious malaria, by 2009, China has been effectively controlled the rise of vivax malaria epidemic and outbreak, malaria incidence rate has dropped to the lowest level in history.
China began to implement the "Chinese action plans to eliminate malaria. Malaria elimination goals from 2010, strategies and measures and control stage are very different. In the stage of malaria control, the control objective is to reduce the incidence of malaria, and in the elimination of malaria elimination target stage, there is no local infection of malaria cases, therefore, timely and accurately find every possible source of infection is the key to blocking the spread of malaria. Malaria elimination guide in malaria elimination stage > requirements for all suspected cases of malaria in laboratory. But with the malaria incidence decreased in patients with clinical blood parasite density decreased, low proportion of clinical malaria parasite density rise, bring new challenges to the laboratory diagnosis of malaria blood smear. The traditional is the most common method for laboratory diagnosis of malaria, but the sensitivity and accuracy of examination Examiners rely on technology and experience, and the low parasite density detection sensitivity is not high. The development of new technology for rapid diagnosis of malaria (RDTs) has the advantages of simple and fast, but the existing RDTs detection rate of low parasite density infection, especially the detection sensitivity of Plasmodium vivax nested PCR is often low. Gene detection technology has the advantages of high Gao Min sensibility, specificity, but it requires special equipment and reagents, not only the price is more expensive, and the operation steps are complex, it is necessary to carry out detection in central laboratory conditions.
Loop mediated isothermal amplification (LAMP) is a new genetic test, Gao Min has high specificity and sensitivity, simple operation and advantages. In the presence of with strand displacement function of Bst DNA polymerase, after an isothermal amplification step can be completed in a short period of time, can realize the amplification of 109-1010. And produce a kind of magnesium pyrophosphate derivatives, the reaction liquid showed visible white haze, according to reaction liquid turbidity and determining result. In LAMP reaction with calcein and Green nucleic acid dye SYBR I can improve the observation effect. But the lack of sensitivity of calcein, SYBR Green I has inhibitory effect on need to join in the amplification reaction, after the reaction, however, the efficiency of LAMP, leading to a significant risk response after opening the cover are amplified product contamination.
To solve the above problems, according to a kind of visual observation results can vivax LAMP detection method developed by our lab, before joining Green SYBR in amplification of I dye containing microcrystalline wax pill, and after the completion of the reaction can release the dye by heating to melt, the reaction product was seen to the naked eye. At the same time in the test tube to the surface of Lawan the formation of solidification barrier, reaction products can prevent pollution. But only LAMP the visual detection of Plasmodium vivax infection, detection stability also needs to be further improved. Considering the infection of Plasmodium vivax malaria cases in China are mainly imported, out of Plasmodium falciparum, Plasmodium vivax infection of migrant workers, three of Plasmodium vivax and oval Plasmodium cases are increasing year by year, and the vast majority of malaria cases have occurred in the remote areas of poverty, but also an urgent need to develop Detection of four species of Plasmodium, stable, sensitive, specific, simple operation, low cost and new technology to be able to carry out large-scale detection for malaria detection. This study was improved and optimized based on the visualization of LAMP, applied to a variety of parasite detection, at the same time will improve the visualization of LAMP reagent and city the sale of LAMP reagent kit cost-effectiveness to evaluate the possibility of site promotion, and through the test to the sample to evaluate its application value. The research includes three parts:
Improvement of Plasmodium technique by visual loop mediated isothermal amplification (LAMP)
Methods: according to the 18S rRNA gene and mitochondrial gene of Plasmodium genus specific conserved region online design primers, primers and reported in the literature were selected and compared. The reaction temperature, the reaction system of dNTPs concentration, MgSO4 concentration, inner primer concentration were optimized. The blood and filter paper blood samples were extracted by DNA Kit and simplified the heat treatment method of sample DNA extraction, amplification effect visualization technology assessment of LAMP improved treatment on different methods of sample. With Plasmodium falciparum, Plasmodium vivax, three Plasmodium vivax, specific Plasmodium falciparum blood DNA as template to evaluate the visualization technology of LAMP detection of Plasmodium is improved. The plasmid concentration gradient and blood sample simulation gradient the parasite density sample as the template, sensitivity of visual LAMP technology improved evaluation.
Results: the 18S rRNA gene and mitochondrial gene of Plasmodium genus specific conserved region of online designed 8 pairs of primers, through comparison and selection, selection of Pg-18S rRNA primers reported in the literature is the most suitable primers. The reaction system and the reaction conditions have been optimized to improve the visualization of LAMP is: 20mM Tris-HCl pH8.8,10mM KCl 10mM (NH4) 2SO4,0.1%Tween20,8U Bst DNA 1.4mMdNTPs, 9mM MgSO4,0.2 polymerase, M F3 and B3,2.5 M and FIP BIP, 0.8 M LPF and 1 L LPB, DNA template, LAMP and visual detection of Plasmodium DDW to 20 DEG C L64 60min. improved the reaction assay were detected in four different Plasmodium; different methods of processing samples have good amplification effect; the minimum detectable Plasmodium 18S rRNA gene plasmid DNA 3.1 * 100copy/ L, amplification of Tt with the increasing concentration of the plasmid in accordance with the relevant relation of decline; simulation The whole blood sample of the density Plasmodium Plasmodium density can detect the whole blood samples of 4.863p/ mu L.
Two, the cost effectiveness comparison of the visual LAMP and the marketed LAMP Kit
Methods: to detect the parasite samples by LAMP kit and LAMP visualization of commercially available self prepared reagents, the reaction efficiency of two kinds of LAMP detection methods, compare the detection results of sensitivity and specificity, and the reagent cost of two detection methods were compared on visual LAMP technology a new method for price the detection of Plasmodium falciparum in possibility to promote the use of the site.
Results: LAMP reaction kit new visual LAMP technology and procurement for high specificity for detection of Plasmodium, but new visual LAMP amplification efficiency is more excellent and stable; the sensitivity of visualization optimization of LAMP (4.863p/ L) ten times higher than the sensitivity of the 48.63p/ detection kit of LAMP L; visualization LAMP detection of Plasmodium homemade reagent detection of single sample reagent cost is about 7 yuan / share, LAMP reaction kit costs about 166 yuan / share, the former is about 1/24. of the latter
Three, application evaluation of visual loop mediated isothermal amplification for detection of malaria site samples
Methods: the blood samples were detected by filter paper LAMP visualization technology, and the results of the examination results were compared and analyzed, the inconsistent results were microscopic review, review of PCR gene nested DNA concentration detection, sample survey and case review, evaluation of new technology of visual detection of Plasmodium LAMP the application value of malaria elimination stage in our country.
Results: using the new detection technology of visualization LAMP clinical acquisition network reported malaria cases filter paper blood samples of 200, detection of Plasmodium positive of 186 samples, 14 samples were negative, and the microscopic examination results were consistent with all samples with 96.5%. and microscopic examination results by nested PCR review results and test results of LAMP a consistent.3 detected by LAMP and nested PCR positive review, but microscopic examination for negative samples by the provincial examination expert examination review after 1 for vivax malaria positive, but parasite density is very low (16p/ L); the other 2 were by the microscopic examination review still negative samples by means of epidemiological case tracking the investigation, confirmed imported cases of falciparum malaria, and in blood smear before were treated with anti malarial drugs; 4 LAMP detection and nested PCR review was negative but positive samples for microscopic examination by the provincial examination examination review experts were positive, but the kind of DNA concentration detection found no extraction of DNA.
conclusion
1, in this study, Plasmodium specific 18S rRNA primers were selected to establish a visualized LAMP detection technique for detecting four Plasmodium species, and the detection sensitivity reached 4.863p/ L..
2, compared with the commercially available LAMP reaction kit, the two new methods for detection of Plasmodium falciparum were all highly specific, but the efficiency and sensitivity of the new visualization LAMP amplification were significantly higher than those of the commercially available LAMP LAMP. The cost was only 1/24. of the commercially available LAMP reaction kit.
3, the new visual LAMP detection technology shows that the detection results of filter paper blood samples collected at the scene show higher sensitivity than microscopy. It can be used for the detection of low protozoan density and the blood samples taken from antimalarial drugs which are difficult to detect by routine microscopy.
4, the new visualization LAMP detection technology has the advantages of sensitivity, efficiency, simplicity, rapidness and mass detection. It can be used as a new tool for gene detection of Plasmodium, and it is widely applied in the elimination stage of malaria in China.

【学位授予单位】：江苏省血吸虫病防治研究所
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R440;R382.31

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