【作者】 向文龙；

【导师】 胡贵方；

【作者基本信息】 南方医科大学 ， 流行病与卫生统计学， 2010， 硕士

【摘要】 研究背景人星状病毒(Human Astrovirus, HAstV)首次于1975年由Appleton和Higgins用电镜检测胃肠炎患儿粪便标本时发现。星状病毒属单独的星状病毒科,星状病毒属,无衣壳单股正链RNA病毒。病毒颗粒直径约28 nm,由于电镜下病毒颗粒呈星形,因此被Madeley和Cosgrove命名为星状病毒。病毒基因全长618 kb,包含有三个开放阅读框架(open reading frames):ORF1a、ORF1b、ORF2。ORF1a、ORF1b在5’端,为高度保守区域,主要编码蛋白酶及RNA多聚糖；ORF2在3’端,基因长度为2388 kb,为多变区,该区域主要编码衣壳蛋白；此外,基因组还包括一个5’非编码区与一个3’端非编码区及一个约30个核苷酸的多聚核苷酸尾。HAstV是引起婴幼儿腹泻的最主要的病原之一,对志愿者和自然感染者的研究均证实它与腹泻密切相关。世界各地均已有HAstV感染的报道,既可散发也可以引起暴发流行,亦可引起医源性感染。与轮状病毒相似,HAstV感染多发生在2岁以内尤其是1岁以内的婴幼儿。住院腹泻患儿中HAstV检出率为2.5%-9.0%,目前被认为是婴幼儿病毒性胃肠炎的第二位病因,仅次于轮状病毒。此外,老年人和免疫缺陷患者也是HAstV感染的高危人群,人类免疫缺陷病毒感染者、接受骨髓移植及联合免疫缺陷患者发生该病毒感染均已有报道。HAstV亦是健康成年人发生胃肠炎的病因之一。应用多克隆抗体和单克隆抗体,可将HAstV划分为8个血清型。HAstV广泛分布于世界各地,各血清型流行情况因地区和流行年不同而有所不同,其中多以血清型1型为主。HAstV感染具有较明显的季节性,一般在温带地区的流行季节为冬季,而在热带地区的流行季节则为雨季。HAstV血清流行病学的研究显示,5岁儿童抗1型HAstV的IgG抗体阳性率达90%,说明儿童5岁以前对HAstV已有广泛的接触。人们先后从猫、羊、猪等动物粪便中及淤泥、湖水中分离出了星状病毒,Miossee报道曾从甲壳类水生物中分离出该病毒,提示星状病毒有可能像甲型肝炎病毒一样通过甲壳类水生物传播。简便、快速地检测HAstV,一直是人们研究的课题。目前检测HAstV常用的方法有：1.电镜法(electronic microscope):Appleton等于1975年首次用电镜发现了HAstV,此后十多年时间里,电镜是检测该病毒的主要方法。此方法形象、直观,但仅有10%的星状病毒具有典型的星形外观,故敏感性较低。若在标本中加入荧光标记的抗体,可提高检出率,称为免疫电镜法。当病毒滴度低时,免疫电镜法仍有较高的假阴性,且由于价格昂贵,技术条件要求高,不适合大规模的流行病学调查。2.细胞培养(cell culture)和病毒分离(virus separate):1977年Lee和Kurtz采用人胚肾细胞成功分离了HAstV。1990年Willcocks等采用传代细胞系人类结肠癌细胞(CaCo-2)直接从腹泻标本中分离出HAstV,且HAstV于CaCo-2细胞内增殖良好,这使HAstV的检测方法发生了里程碑的进步。人们可利用增殖的病毒制备不同血清型的单克隆抗体,这为以后应用酶免疫法进行大量流行病学调查奠定了基础,正因为如此,星状病毒腹泻才逐渐为大家所认识和重视。3.酶免疫法(enzyme immunoassay, EIA)及酶联免疫吸附试验(enzyme linked immunosorbent assay, ELISA):Herrmand等在成功制备HAstV单克隆抗体的基础上建立了EIA和ELISA,该法操作更简单,具有更好的敏感性及特异性,并能进行分型。但用于检测的单克隆抗体在商业上往往很难获得且价格昂贵,故作为常规检测方法在目前有较大的困难。4.逆转录聚合酶链式反应(RT-PCR):随着对星状病毒培养、分离及测序的成功,星状病毒的RT-PCR检测法也就应运而生。从1993年开始,RT-PCR就应用于检测星状病毒,并与EIA进行了对比,发现RT-PCR法比EIA有更高的敏感性及特异性,不受难以获得的血清抗体的限制,故目前大多数星状病毒的报道均是用该法进行研究。5.血清学检测：HAstV血清学检测方法包括免疫电镜(IEM)、放射免疫法(RIA)、免疫荧光法(IFA)、酶免疫法(EIA)等。血清学检测方法可帮助了解HAstV感染状况,目前认为其特异性抗体仅具有部分保护作用,故现在开展得较少。环介导等温扩增技术(loop-mediated isothermal amplification, LAMP)是由日本荣研株式会社的Notomi等2000年开发出来的一种新型的核酸扩增方法。LAMP的反应原理是利用Bst大片段DNA聚合酶和根据不同靶序列设计的内引物(FIP由Flc和F2组成；BIP由B1c和B2组成)和外引物(F3和B3),特异地识别靶序列上的6个独立区域,在60-65℃恒温条件通过循环链置换反应,形成含有若干交替重复的茎环结构的DNA。LAMP法利用酶反应系统直接扩增靶核酸序列,能使靶核酸序列呈指数级扩增,在45-60min的时间里可达到109-1010数量级；反应过程不需控制温度的变化,在恒温水浴加热即可进行,省去了昂贵的热循环仪的费用。由于两对引物在靶基因上的6个不同部位完全匹配才能进行扩增,所以LAMP法具有很高的特异性。在DNA合成时,从脱氧核酸三磷酸基质(dNTP)中析出的焦磷酸根离子与反应溶液中的镁离子反应,产生大量焦磷酸镁白色沉淀,因此可以把浑浊度作为反应的指标,仅用肉眼观察白色浑浊沉淀就能鉴定扩增与否,而不需要繁琐的电泳和紫外观察。有时,通过肉眼观察白色沉淀来判断会存在一定的误差,日本荣研株式会社利用LAMP反应过程中产生焦磷酸镁白色沉淀,研制出专门用于LAMP检测的实时浊度仪,以实现对LAMP扩增过程的实时监控,使扩增和检测同时完成。此外,还可通过添加荧光染料,如溴化乙锭(EB)、SYBR GreenⅠ等进行染色,来检测扩增是否发生。LAMP反应的最终扩增产物是分子量大小不等的茎环DNA组成的混合物,在2%的琼脂糖凝胶上电泳呈现的是典型的梯状条带。LAMP法由于具有操作简便易行、特异性高、结果判断简单等多方面的优点,故本法一经面世就被应用到很多方面,包括人类、动物和植物致病微生物的检测、动物胚胎性别的鉴别和转基因食品的检测等。研究目的建立一种快速检测人星状病毒1型的环介导等温扩增方法。研究方法1.标本的收集1份HAstV-1型和1份札幌病毒GⅡ型阳性粪便标本来源于中国疾病预防控制中心病毒病预防控制所,2份诺如病毒GⅡ型、3份轮状病毒A组阳性粪便标本由本实验室保存,80份腹泻患者粪便标本于2006年8月至2007年1月期间收集于江门市妇幼保健医院儿科门诊,以及江门市人民医院急诊科和腹泻门诊。收集的粪便标本用PH7.2的PBS配制成悬液保存于-40℃。2.引物设计针对编码非结构蛋白的基因组保守区域ORF1a,通过ClustalW2在线软件比对GeneBank上已公布的ORFla序列确定LAMP目标序列,输入在线软件Primer Explorer V4设计出特异的RT-LAMP引物,其序列(5’-3’)为：F3:GCATTATCTTCTTGTGCTTCA; B3:TCACGGATCTCGAACCTG;FIP:CCACCAGCAATTAATACTGCTGTAGGAAGACTCCAACTATGTGAGC;BIP:GCACGACCACGTCATTGTTTCAATGAAAACAGTTGCCATAC.3.RNA的提取用Trizol法从收集的粪便标本提取RNA并溶于DEPC处理水,测定其230nm、260 nm、280 nm和310 nm的OD值,保存于-80℃。4. RT-LAMP条件优化、产物的检测和酶切鉴定反应体系置60℃恒温反应45min、60min、75min、90min,最后80℃孵育5min以灭活酶终止反应。产物通过凝胶电泳后溴化乙锭染色紫外线下观察,以及荧光染料SYBR Green I染色后肉眼观察。对RT-LAMP产物进行切胶、称重,经凝胶回收试剂盒纯化回收后用限制性内切酶TaaI进行切割,凝胶电泳观察酶切片段。5. RT-PCR和RT-LAMP检测HAstV-1型的特异性、敏感性分析5.1以RT-LAMP的外引物F3、B3作为RT-PCR的上、下游引物进行RT-PCR扩增,对其产物进行切胶、称重,经凝胶回收试剂盒纯化回收后,与pMD18-T载体连接,转化至感受态大肠杆菌DH5a。通过蓝白斑筛选试验挑出白色菌落接种到含Amp的LB液体培养基震荡培养,再提取质粒DNA作模板用引物F3、B3进行PCR扩增来鉴定筛选正确的克隆。测定质粒溶液230 nm、260 nm、280nm和310 nm的OD值,将浓度为2.3×108copies/μl质粒DNA溶液保存于-20℃备用。5.2 1份HAstV-1型、1份札幌病毒GⅡ型、2份诺如病毒GⅡ型、3份轮状病毒A组阳性粪便标本作为检测对象,提取RNA同时进行RT-LAMP和RT-PCR检测。5.3 HAstV-1型阳性标本所提取RNA的一系列稀释度为：1ng/μl, 100pg/μl, 10pg/μl,1pg/μl,100fg/μl,10fg/μl,分别作模板用RT-LAMP和RT-PCR检测。5.4质粒DNA浓度按10×梯度稀释,其浓度为2.3×107copies/μl到23copies/μl,并作模板用LAMP和PCR检测。6.检测临床腹泻患者粪便标本80份非细菌性腹泻患者粪便标本提取RNA后,同时用RT-LAMP和RT-PCR检测。研究结果1.各反应时间的RT-LAMP产物电泳呈特征性梯状条带,其中90min产物条带较明显,以90min为最佳反应时间。2. RT-LAMP产物加入荧光染料,管中反应液变绿,呈阳性结果；阴性对照管和空白对照管保持橙色,呈阴性结果。3.用限制性内切酶Taal切割其产物,结果显示酶切片段与预期值(206,225,244 bp)相符。4. RT-LAMP和RT-PCR分别检测HAstV-1型均呈阳性,而其他病毒株均呈阴性。5. RT-LAMP检测HAstV-1型RNA下限为5 pg/管,LAMP检测重建质粒的下限为230 copies/管；RT-PCR和PCR则为50 pg/管和2.3×103 copies/管。6. RT-LAMP和RT-PCR分别检测80份临床腹泻患者粪便标本,其中3份均呈阳性,其余均为阴性。两种方法检测结果一致。结论本研究建立的RT-LAMP方法检测HAstV-1型的特异性和敏感性较好,且反应耗时较短,实用性强,适合于基层实验室和现场的应用。更多还原

【Abstract】 BackroundHuman Astrovirus (HAstV) was discovered for the first time in 1975 by Appleton and Higgins when they were using electron microscope for the detection stool samples of children with gastroenteritis. Astrovirus is a non-capsid single-strand sensitive RNA virus and a family of Astroviridae and Astrovirus. The diameter of the viral particle is 28nm, which shows a shape of star under electron microscope, therefore, the virus was named as astrovirus by Madeley and Cosgrove. The viral genome is 618 kb in length, containing three open reading frames:ORFla, ORFlb, ORF2. ORF1a and ORF1b are at the 5’end, which are highly conserved regions, mainly encoding the protease and RNA polysaccharide; ORF2 is at the 3’end with a length of 2388 kb, which is a ever-changing area, mainly encoding the capsid protein. In addition, the genome also includes a 5’non-coding region, a 3’non-coding region and an approximately 30-nucleotide poly-nucleotide tail.HAstV is one of the most important pathogens that cause diarrhea in infants, as it was proved that astrovirus closely related to diarrhea by the study of volunteers and natural infection. Astrovirus infection was reported around the world, which could be found separately or in an epidemic outbreak and also in iatrogenic infections. Astrovirus infection occurs in infants less than 2 years of age, especially infants less than 1 year of age, which is similar to rotavirus. With a detection rate from 2.5% to 9.0% in hospitalized children with diarrhea, astrovirus is now considered as the second cause of viral gastroenteritis in infants and young children, second only to rotavirus. In addition, the elderly and patients with immunodeficiency are also at high risk. Astrovirus infection was reported in human immunodeficiency virus infection, bone marrow transplantation and combined immunodeficiency patients. Astrovirus is also one of the causes of gastroenteritis occurred in healthy adults.With the application of polyclonal antibodies and monoclonal antibodies, astrovirus can be divided into eight serotypes. Astrovirus widely distributed around the world and the prevalence of its serotypes varies in regions and years, mostly dominated by serotype 1. With an apparent seasonality, astrovirus infection generally occurs in the temperate regions in winter, compared with the tropical regions in rainy season. Serum epidemiological studies have shown a positive rate of 90% of IgG antibody for serotype-1 astrovirus in 5-year-olds, indicating that children have had extensive contacts with astrovirus before the age of five. Afterwards people have isolated astrovirus from the cats, sheep, pigs and other animal feces, and mud, the lake water. It has been reported Miossee isolated astrovirus from the aquatic crustacean, suggesting that astrovirus may be the same as the hepatitis A virus spreading through the aquatic crustaceans. To detect astrovirus simply and quickly has been one subject of study.Now commonly used detection methods are as follows:1. Electron microscopy. Appleton, etc. discovered astrovirus with electron microscope for the first time in 1975, and since then in more than ten years, the electron microscopy was the primary means for the detection of astrovirus. It is visualized and intuitive by this method, however, only 10% of astrovirus has a typical star-shaped appearance and therefore it is less sensitive. Adding fluorescent labeled antibodies to samples can improve detection rate, which is known as immune electron microscopy. However, when the virus titer is low, there is still a high false negative, and it is expensive, demanding better technical conditions, so it is not suitable for large-scale epidemiological investigations.2. Cell culture and virus separation. In 1977, Lee and Kurtz successfully separated astro virus by using human embryonic kidney cell. In 1990, Willcocks, etc. directly isolated astrovirus from the diarrhea samples by using cell lines with passage of human colon cancer cells (CaCo-2) and astrovirus proliferated well in CaCo-2 cell, which made great progress in astrovirus detection methods. People could make use of the proliferated virus to produce monoclonal antibodies of different serotypes, and monoclonal antibodies laid the foundation for the subsequent enzyme-linked immunosorbent method applying for a large number of epidemiological investigations, and as such, gradually diarrhea caused by astrovirus was known and gained attention.3. Enzyme immunoassay (EIA) and enzyme-linked immunosorbent assay (ELISA):Based on the successful preparation of monoclonal antibodies of astrovirus, Herrmand, etc. developed the method of EIA and ELISA, which was simpler with better sensitivity and specificity and could carry out genotyping. However, because monoclonal antibodies for the detection in the business were often difficult to obtain and expensive, there was great difficulty for conventional application at present.4. Reverse transcription-polymerase chain reaction (RT-PCR). With the success of the cultivation, separation and sequencing of astrovirus, RT-PCR assay also emerged. Beginning in 1993, RT-PCR assay was applied for the detection of astrovirus, and a comparison with the EIA found that RT-PCR assay had higher sensitivity and specificity than the EIA getting rid of restrictions of serum antibodies that were difficult to obtain. Therefore, at present this method was used in most reported studies of astrovirus.5. Serological tests. Serological detection methods of astrovirus include immuno-electron microscopy (IEM), radioimmunoassay (RIA), immunofluorescence assay (IFA), enzyme immunoassay (EIA) and so on. Serological detection methods may help to know astrovirus infection, and because specific antibodies of astrovirus were now considered to only have some protective effect, it was seldom carried out.Loop-mediated isothermal amplification (LAMP) is a new type of nucleic acid amplification method developed in 2000 by Notomi, etc. of Eiken Chemical Co., Ltd., in Japan. The reaction principle of LAMP is to make use of Bst large fragment DNA polymerase, inner primers (FIP formed by F1c and F2; BIP formed by B1c and B2) and outer primers (F3 and B3) which are designed according to different target sequences and specifically identify target sequences on the six separate regions, through the strand displacement cycling reaction in the constant temperature raging from 60 to 65 centi-degree, to generate DNA product containing a number of alternative repetitive stem-loop structure.The enzyme reaction system is used in LAMP, directly amplifying the target nucleic acid sequence, enabling the exponential amplification of the target nucleic acid sequence that reaches 109 to 1010 order of magnitude in 45 to 60 minutes; during the reaction process it is no need to control the temperature changes for the reaction can be carried out by heating with constant temperature in water bath, eliminating the costs for expensive thermal cycler. As amplification proceeds only when two pairs of primers exactly match the six different regions on target gene, LAMP has a high specificity. In DNA synthesis, the pyrophosphate ions separated from deoxynucleic acid triphosphate substrate (dNTPs) react with the magnesium ions in the reaction solution, producing a large number of white precipitate of magnesium pyrophosphate. Therefore, turbidity can be used as reaction indicator, as observing a white cloudy precipitate only by the naked eyes will be able to identify whether the amplification occurs or not, without the need for tedious agarose gel electrophoresis and observation under UV. Sometimes, there may be some errors to judge by observing a white precipitate with the naked eyes. The Eiken Chemical Co., Ltd. made use of the white precipitate of magnesium pyrophosphate produced in LAMP reaction and developed a real-time turbidimeter specially for the detection of LAMP reaction, in order to accomplish amplification and detection of LAMP simultaneously with real-time monitoring during the process. In addition, it is also useful to add fluorescent dye, such as ethidium bromide (EB), SYBR GreenⅠdye etc., to detect amplification. The final product of LAMP reaction is composed of a mixture of stem-loop DNA in different sizes, and in 2% agarose gel electrophoresis shows a typical DNA ladder. Since LAMP has many advantages such as being easy to perform with high specificity and being simple to determine the result, once this method is available it has been applied for the many aspects, including detection of pathogenic micro organisms in human, animal and plant, identification of gender of animal embryos and detection of genetically modified food.ObjectiveTo establish a loop-mediated isothermal amplification method for rapid detection of human astrovirus of serotype 1.Methods1. Collection of samplesOne stool sample positive for HAstV genotype 1 and one for sapovirus genotypeⅡwere obtained from institute of viral disease control and prevention, Chinese center for disease control and prevention. Two stool samples positive for norovirus genotyopeⅡand three for rotavirus group A were preserved in the lab. Eighty stool samples from patients with nonbacterial diarrhea were collected from August 2006 to January 2007 in pediatric clinic of Jiangmen maternal and child health hospital and emergency department and diarrhea clinic of Jiangmen people’s hospital. Stool samples were preserved as suspensions with PBS of PH7.2 under-40 centi-degree.2. Primer designTargeting at the conserved region of ORF1a on the genome encoding the non-structural protein, the target sequence of LAMP was identified with alignment of sequences of HAstV ORFla published on the GeneBank using the software ClustalW2. Specific RT-LAMP primers was designed by entering the target sequence into the online software Primer Explorer V4, and the nucleic acid sequences of primers (from 5’to 3’) were as follows:F3:GCATTATCTTCTTGTGCTTCA; B3:TCACGGATCTCGAACCTG;FIP:CCACCAGCAATTAATACTGCTGTAGGAAGACTCCAACTATGTGAGC;BIP:GCACGACCACGTCATTGTTTCAATGAAAACAGTTGCCATAC.3. RNA preparationRNA was extracted from stool specimens collected by the Trizol method and dissolved in DEPC treated water, with the OD values at 230 nm,260 nm,280 nm and 310 nm determined and kept at-80 centi-degree until used.4. Optimization of RT-LAMP reaction condition and detection and digestion of RT-LAMP productThe reactions were completed at a constant temperature of 60 centi-degree within a duration of 45,60,75 and 90 minutes, respectively, with a final incubation at 80 centi-degree for 5’minutes to inactivate enzymes and terminate the reaction. Products can be observed under ultraviolet light by gel electrophoresis and ethidium bromide staining, and with the naked eye after the fluorescent dye SYBR Green I staining. RT-LAMP products were purified using a gel extraction kit. After the product was digested by the restriction enzyme Taal, fragments produced by digestion could be detected by agarose gel electrophoresis.5. Detection specificity and sensitivity of HAstV-1 in stool samples by RT-PCR and RT-LAMP5.1 The outer RT-LAMP primers F3 and B3 was used as the forward and backward primers for RT-PCR amplification. RT-PCR products were purified using a gel extraction kit and ligated into plasmid pMD18-T vector and the recombinant plasmid was transformed into Escherichia coli strain DH5a. By blue-white colony selection the white colonies were picked for amplification with shaking in LB broth medium containing ampicillin. The plasmid was extracted as template for PCR by F3 and B3 primers set to identify the correct clone. The OD values of plasmid solution at 230 nm, 260 nm,280 nm and 310 nm were determined and plasmid solution with a concentration of 2.3×108copies/μl were kept at-20 centi-degree until used.5.2 One stool sample positive for HAstV-1, one for sapovirus GII, two for norovirus GII and three for rotavirus group A were detected by RT-LAMP and RT-PCR after RNA extraction.5.3 The stock solution of RNA extracted from positive sample of HAstV-1 was 10-fold serially diluted and the concentrations were 1ng/μl, 100pg/μl, 10pg/μl, 1pg/μl, 100fg/μl and 10fg/μl. RT-LAMP and RT-PCR were used for the detection.5.4 The stock solution of recombinant plasmid was 10-fold serially diluted from 2.3×107 copies/μl to 23 copies/μl. LAMP and PCR were used for the detection.6. Evaluation of RT-LAMP and RT-PCR using clinical samplesAfter RNA extraction 80 stool samples collected from patients with non-bacterial diarrhea were detected by RT-LAMP and RT-PCR.Results1. In different reaction time the RT-LAMP products showed the characteristic ladders by agarose gel electrophoresis, with the most apparent ladders observed in 90 minutes which was considered to be the optimal reaction time.2. By adding fluorescent dye into RT-LAMP products, the reaction solution turns green, showing the positive result while negative control and no template control keep the color of orange, showing the negative results.3. After digestion by the restriction enzyme Taal, it showed that fragments produced by digestion have the expected sizes (206,225,244 bp).4. RT-LAMP and RT-PCR were used to detect HAstV-1 and positive results were showed, while other viral strains were negative.5. The lower limit of RT-LAMP for the detection of HAstV-1 RNA was 5 pg per reaction and the lower limit of LAMP for the detection of recombinant plasmid was 230 copies perreaction while RT-PCR and PCR was 50 pg per reaction and 2.3×103 copies per reaction, respectively.6. RT-LAMP and RT-PCR were used to detect 80 stool specimens collected from patients with diarrhea, of which three were positive and the rest were all negative. The test results of two methods were consistent.ConclusionThe RT-LAMP assay established in this study, with high specificity and sensitivity for the detection of HAstV-1 and less time cost, was practical and suitable for primary laboratory and field applications.更多还原