本文選題：手足口病 + EV71��； 參考：《南方醫(yī)科大學(xué)》2012年博士論文

【摘要】：手足口病(hand,foot,mouth disease, HFMD)是以發(fā)熱和手、足、口腔等部位出現(xiàn)皮疹、皰疹為特征的兒童常見(jiàn)病。絕大部分病情較輕,少數(shù)可發(fā)生嚴(yán)重神經(jīng)系統(tǒng)并發(fā)癥并致相當(dāng)數(shù)量?jī)和劳?是中國(guó)及亞洲的主要公共衛(wèi)生問(wèn)題之一。 HFMD由人類腸道病毒(human enterovirus, HEV)感染引起。其中腸道病毒71型(EV71)是最重要的病原體,因其更易導(dǎo)致神經(jīng)系統(tǒng)感染甚至死亡。EV71屬小RNA病毒科,成熟EV71病毒顆粒外殼由60個(gè)拷貝的P1蛋白組成,P1又分為VP1-VP4四種衣殼蛋白。按照VP1區(qū)不同EV71分A、B、C三個(gè)基因亞型,中國(guó)大陸流行C4亞型。臨床實(shí)驗(yàn)室HFMD病原學(xué)診斷以分子生物學(xué)和血清學(xué)檢測(cè)為主。目前,尚無(wú)有效的抗病毒藥物和疫苗防治EV71感染。對(duì)于EV71外殼抗原結(jié)構(gòu)、中和性表位及其在病毒致病機(jī)制中的作用的認(rèn)識(shí)還很有限。已證實(shí)VP1、VP2、VP3具中和活性抗原位點(diǎn),VPl是最主要的抗原決定簇,VP4雖位于病毒表面內(nèi)側(cè),但能產(chǎn)生相應(yīng)抗體,推測(cè)其可能參與抗體依賴感染增強(qiáng)效應(yīng)(ADE)而加強(qiáng)病毒對(duì)機(jī)體的攻擊。 本研究對(duì)廣東地區(qū)發(fā)生的HFMD進(jìn)行病原學(xué)監(jiān)測(cè)：收集了2009-2011年EV71流行株及所致疾病的臨床特征、基因分型及其來(lái)源、變遷及突變等信息,為控制和預(yù)防HFMD提供預(yù)警及對(duì)策的參考資料；采用多種目前實(shí)驗(yàn)室檢測(cè)病毒的方法對(duì)臨床常用的捕獲ELISA發(fā)檢測(cè)抗EV71-IgM用于EV71病原診斷進(jìn)行評(píng)估,使臨床更合理使用；在此基礎(chǔ)上,采用載體pQE30a和大腸桿菌M15系統(tǒng)重組表達(dá)了EV71臨床分離株的VP1-4蛋白,通過(guò)免疫小鼠獲得針對(duì)這些蛋白的單克隆抗體,為研究EV71病毒衣殼的抗原結(jié)構(gòu)及功能、揭示EV71致病機(jī)制以及疫苗制備提供實(shí)驗(yàn)依據(jù)。本研究包含三個(gè)部分內(nèi)容： 第一部分2009-2011年EV71致手足口病臨床及病原學(xué)特征 2009～2011年來(lái)自廣東各地在我院就(轉(zhuǎn))診的臨床診斷HFMD(含皰疹性咽峽炎)分別有107、553、579例,實(shí)時(shí)熒光RT-PCR對(duì)腸道病毒通用型核酸檢出率分別為87.9%(94/107)、91.2%(505/553)和88.8%(515/579),EV71占所有病原的比例分別為28.1%(30/107)、45.6%(252/553)和38.7%(221/579),顯示EV71仍為廣東手足口病最主要病原。 1239例患者中男性813例,女性426例,男女比1.91,EV71感染性別比與全部EV感染無(wú)差異(P=0.765)�；颊吣挲g最小1個(gè)月,最大35歲,中位數(shù)分別是2.08歲、2.58歲和2.41歲；1歲和2歲是發(fā)病最多的兩個(gè)年齡段,占73.24歲以下兒童占患者總數(shù)85.0%；6個(gè)月以下發(fā)病者21例,EV71感染8例；各年齡段感染EV71與EV感染比例相似。診斷為重癥手足口病81例,年齡最小4個(gè)月,最大10歲,中位數(shù)2.0歲,1～2歲是出現(xiàn)重癥最多的年齡段,各年齡段發(fā)生重癥手足口病例與普通手足口病例相似；男女比：2.24:1,略(?)性別比,但無(wú)統(tǒng)計(jì)學(xué)差異(P=0.62),提示重癥并不傾向于男孩。 監(jiān)測(cè)顯示2009-2011年廣東以散發(fā)流行為主。每年出現(xiàn)兩個(gè)發(fā)病高峰：初夏(5、6月間)和秋季(10、11月),2010年5月中旬達(dá)最高峰,持續(xù)約1個(gè)月,10月出現(xiàn)小高峰；2011年發(fā)病高峰期推遲至半個(gè)月,春夏季EV71導(dǎo)致的HFMD較秋季略高。 臨床表現(xiàn)以手掌、臀部、足底斑疹,口腔皰疹為主：90%上的(?),60%以上發(fā)病初期有發(fā)熱伴出疹,順序不一。少數(shù)(7.2-11.3%)表現(xiàn)呼吸道感染癥狀。極少數(shù)出現(xiàn)神經(jīng)性癥狀,如嘔吐、抽搐、驚跳或頭痛等。EV71感染臨床表現(xiàn)與EV所致HFMD相似,無(wú)統(tǒng)計(jì)學(xué)差異。2010～2011年分別有89和144例患兒經(jīng)ICU治療,其中EV感染分別是75例(14.9%)和128例(24.9%)。EV71感染較EV感染更多需要ICU治療(P值分別為0.037和0.000)。所有診斷為重癥手足口病者均有腸道病毒相關(guān)性腦病發(fā)生,如病毒性腦炎等,呼吸道并發(fā)癥主要是神經(jīng)源性肺水腫。 擴(kuò)增測(cè)序33株2009-2010年臨床分離EV71(2009年5株,2010年28株)的部分VP1區(qū),經(jīng)比對(duì)分析,確定均屬C4a亞型,與廣東歷年分離毒株序列比較未見(jiàn)明顯差異；與2008年阜陽(yáng)EV71流行株比較亦未見(jiàn)明顯差異,表明為國(guó)內(nèi)循環(huán)流行株；與臺(tái)灣近年流行株的基因亞型不同,表明兩岸毒株進(jìn)化來(lái)源不同。通過(guò)Clustal W軟件對(duì)臨床分離株與標(biāo)準(zhǔn)株BrCr(U22521).臺(tái)灣1998年流行株(Taiwan98)比對(duì)VPl區(qū)序列：核苷酸相似度93.7%-100%,氨基酸相似度97.0%-100%。第22、98號(hào)位氨基酸與臺(tái)灣98年流行株和標(biāo)準(zhǔn)株BrCr略有不同。 第二部分實(shí)時(shí)熒光RT-PCR和EV71-IgM捕獲ELISA法對(duì)EV71致手足口病診斷的評(píng)估 為評(píng)估捕獲ELISA (?)法檢測(cè)EV71-IgM,我們收集了從發(fā)病第1天到158天的134例EV71感染血清256份,發(fā)現(xiàn)發(fā)病第1天即可檢出EV71-IgM,隨發(fā)病天數(shù)增多檢出率升高,第5天達(dá)100%；發(fā)病3個(gè)月后血清EV71-IgM檢出率明顯下降。急性期(發(fā)病7天的(?)檢測(cè)敏感度90.2%(138/153),發(fā)病90天內(nèi)敏感度93.6%(233/249)。 與CA16-IgM檢測(cè)存在交叉反應(yīng)。實(shí)驗(yàn)顯示122份CA16感染血清中38份EV71-IgM陽(yáng)性,49份其它腸道病毒感染血清中14份EV71-IgM陽(yáng)性,105份其它呼吸道病毒感染血清有2份(來(lái)自同一呼吸道合胞病毒A和B型雙重感染患者)EV71-IgM陽(yáng)性對(duì)于其它腸道病毒,EV71-IgM捕獲法ELISA法檢測(cè)特異性為69.6%(52171.95。95(?):65.2-72.4%),對(duì)于其它呼吸道病毒特異性98.1%(2/105,950%可信區(qū)間：96.5-99.5%),檢測(cè)陽(yáng)性預(yù)測(cè)值81.3%(95%可信區(qū)間：76.9-85.1%),陰性預(yù)測(cè)值91.0%(95%可信區(qū)間：87.5-93.8%)。206份EV71感染血清檢出EV71-IgM陽(yáng)性199份(95.7%)、CA16-IgM陽(yáng)性58份(28.2%),其中EV71-IgM的OD值高于CA16-IgM者56份(占96.6%)；119份CA16感染血清CA16-IgM陽(yáng)性83份(69.7%),EV71-IgM陽(yáng)性36份(30.3%),其中CA16-IgM的OD值高于EV71-IgM者33份(占91.7%),實(shí)際感染的病原產(chǎn)生的IgM檢測(cè)OD值更高,可正確區(qū)分大部分感染病原體。 111例同日收集的肛拭子和血清標(biāo)本分別用ELISA和實(shí)時(shí)熒光RT-PCR兩種方法檢測(cè),發(fā)現(xiàn)兩種方法診斷EV71感染無(wú)顯著性差異(McNemar's χ2檢驗(yàn)P=0.648),一致性較好(Kappa值0.729)。 第三部分EV71病毒衣殼蛋白P1單抗的制備 依據(jù)EV71基囚序列信息(FJ194965.1)設(shè)計(jì)VP1～VP4基因全長(zhǎng)序列引物,經(jīng)PCR擴(kuò)增,Kpn Ⅰ和Hind Ⅲ雙酶切PCR產(chǎn)物,連接載體pQE30a,經(jīng)測(cè)序確認(rèn)與原始序列完全一致后將該重組質(zhì)粒轉(zhuǎn)化大腸桿菌M15。經(jīng)誘導(dǎo)表達(dá)VP1～VP4蛋白,均以包包涵體形式存在,以8M尿素變性及梯度復(fù)性,復(fù)性蛋白經(jīng)HisBind Resin層析柱純化.獲得純化VP1～4蛋白。以重組VP4蛋白與純化EV71病毒交替免疫小鼠,經(jīng)小鼠脾細(xì)胞與雜交瘤細(xì)胞融合獲得雜交瘤細(xì)胞克隆,分別采用重組蛋白VP1～4和病毒液包被ELISA、間接免疫熒光法篩選克隆,采用免疫印跡鑒定針對(duì)的靶蛋白。 以FV71病毒液、溶于8M尿素的重組蛋白VP1、VP2和VP4分別包被板條,ELISA檢測(cè)融合細(xì)胞克隆上清,挑選OD值高的克隆,進(jìn)一步間接免疫熒光法對(duì)EV71感染和未感染RD細(xì)胞反應(yīng),篩選僅對(duì)EV71感染RD細(xì)胞有陽(yáng)性熒光的克隆。克隆上清采用純化EV71病毒液和重組VP1-VP4蛋白經(jīng)SDS-PAGE膠分離的蛋白組分進(jìn)行免疫印跡反應(yīng)鑒定抗體針對(duì)的靶蛋白。初步獲得25株針對(duì)P1蛋白的單抗：6株針對(duì)EV71病毒VP1蛋白(天然蛋白),其中4株對(duì)重組VP1蛋白有反應(yīng)；3株僅針對(duì)重組VP2蛋白;2株針對(duì)病毒VPl和VP2蛋白,其中針對(duì)重組VP1和VP2各一株；1株針對(duì)重組蛋白VP4；另有13株目前尚未鑒定出針對(duì)的靶蛋白。 小結(jié) 根據(jù)以上三個(gè)部分的研究成果,本研究小結(jié)如下： 1.2009-2011年監(jiān)測(cè)EV感染手足口病患者病原,EV71感染分別是：30、252和221例,分別占28.1%(30/107)、45.6%(252/553)和38.7%(221/579),EV71仍為廣東手足口病最主要病原。 2.2009-2011年廣東以散發(fā)為主,每年出現(xiàn)一大一小兩個(gè)發(fā)病高峰,即初夏(5、6月之間)和秋季(10、11月)；1、2歲是發(fā)病最多的年齡段,占73.2%,4歲以下兒童占總數(shù)85.0%；EV71感染致重癥手足口病患者年齡分布與此相似,無(wú)統(tǒng)計(jì)學(xué)差異。 3.2009～2010年廣東EV71臨床分離株屬C4a亞型,與廣東歷年分離株和08年安徽阜陽(yáng)流行株序列比較未見(jiàn)差異,為國(guó)內(nèi)循環(huán)流行毒株；與臺(tái)灣近年流行株比較,為不同基因亞型,提示兩地毒株進(jìn)化來(lái)源不同；與標(biāo)準(zhǔn)株BrCr、臺(tái)灣1998年流行株Taiwan98VP1區(qū)核苷酸相似度93.7%-100%,氨基酸相似97.0%-100%。第22、98號(hào)位氨基酸于臺(tái)灣98年流行株和標(biāo)準(zhǔn)株BrCr略有不同。 4.捕獲ELISA法EV71-IgM檢測(cè)診斷EV71致手足口病急性期敏感度為90.2%(138/153),特異性分別為69.6%(52/171,相對(duì)其他腸道病毒)和98.1%(2/105,相對(duì)其他呼吸道病毒),檢測(cè)陽(yáng)性預(yù)測(cè)值81.3%(95%可信區(qū)間：76.9-85.1%),陰性預(yù)測(cè)值91.0%(95%可信區(qū)間：87.5-93.8%)。與實(shí)時(shí)熒光RT-PCR方法診斷EV71感染無(wú)顯著性差異(McNemar's χ2檢驗(yàn)P=0.648),一致性較高。該方法適用于臨床。 5.捕獲ELISA法EV71-IgM檢測(cè)診斷EV71雖然對(duì)CA16感染HFMD血清有交叉反應(yīng)性(28.2%和30.3%),但較高的OD值可以反映感染病毒的類型。 6.采用EV71純化病毒和重組VP4蛋白免疫小鼠,初步獲得25株針對(duì)P1蛋白的單克隆抗體,經(jīng)EV71病毒液、VP1-VP4重組蛋白ELISA、間接免疫熒光篩選和免疫印跡鑒定分別是：針對(duì)VP1蛋白6株,VP2蛋白3株、同時(shí)針對(duì)VP1和VP22株和VP4蛋白1株,尚未確定13株。
[Abstract]:Hand (foot, mouth disease, HFMD) is a common disease in children with fever and hand, foot and mouth and other parts of the mouth. Most of the disease is common in children. The vast majority of the disease is mild. A few serious neurological complications and a considerable number of children are killed. It is one of the major public health problems in China and Asia.
HFMD is caused by the human enterovirus (human enterovirus, HEV) infection. Among them, the enterovirus 71 (EV71) is the most important pathogen, because it is more susceptible to the infection of the nervous system and even the death of.EV71 family in the small RNA virus family. The mature EV71 virus particle shell is composed of 60 copies of the P1 protein, and P1 is divided into VP1-VP4 four capsid proteins. Three subtypes of EV71 A, B, C, and C4 subtypes in mainland China. The diagnosis of HFMD in clinical laboratories is based on molecular biology and serological detection. At present, there are no effective antiviral drugs and vaccines for the prevention and treatment of EV71 infection. The understanding of the antigen structure of EV71, the neutralization epitopes and their role in the pathogenesis of the virus It has been limited. VP1, VP2, VP3 have been proved to have neutralization active antigen sites, and VPl is the most important antigenic determinant. Although VP4 is located inside the surface of the virus, it can produce corresponding antibodies. It is presumed that it may participate in the antibody dependent infection enhancement effect (ADE) to strengthen the attack of the virus to the body.
In this study, the etiology of HFMD in Guangdong was monitored. The clinical features, genotyping, its origin, changes and mutations were collected for 2009-2011 years of EV71 epidemic strains and the disease, and the reference materials for the prevention and control and prevention of HFMD were provided. The use of ELISA hair detection to detect anti EV71-IgM for the diagnosis of EV71 pathogens and to make the clinical use more reasonable. On this basis, the carrier pQE30a and Escherichia coli M15 system were used to restructure the VP1-4 protein of the EV71 clinical isolates, and the monoclonal antibodies against these proteins were obtained by immunizing mice to study the resistance of the EV71 virus capsid. The original structure and function provide an experimental basis for revealing the pathogenic mechanism of EV71 and the preparation of vaccine. This study contains three parts:
Part 1 clinical and etiological characteristics of hand foot mouth disease caused by EV71 in 2009-2011 years
In 2009~2011 years, there were 107553579 cases of HFMD (herpetic angina) from all over Guangdong in our hospital. The detection rate of real time fluorescent RT-PCR for enterovirus general nucleic acid was 87.9% (94/107), 91.2% (505/553) and 88.8% (515/579), and EV71 accounted for 28.1% (30/107), 45.6% (252/553) and 3, respectively. 8.7% (221/579) showed that EV71 is still the most important pathogen of HFMD in Guangdong.
Among the 1239 patients, 813 were male and 426 women were women, and the ratio of male and female was 1.91. The sex ratio of EV71 infection was not different from that of all EV infection (P=0.765). The age was 1 months, the maximum was 35 years, the median was 2.08, 2.58 and 2.41, and 1 and 2 years were the two age groups, which accounted for the total number of patients under the age of 73.24. There were 21 cases of disease and 8 cases of EV71 infection. The proportion of EV71 and EV infection in all ages was similar. The diagnosis was 81 cases of severe hand foot and mouth disease, the minimum age of 4 months, the maximum 10 years, the median 2 years, and 1~2 years of age, similar to those of the ordinary hand foot and mouth cases in each age group; the ratio of men and women: 2.24:1, slightly (?) Sex ratio, but no statistical difference (P=0.62), suggesting that severe illness is not inclined to boys.
The monitoring showed that Guangdong was mainly sporadic in 2009-2011 years. There were two peaks of onset each year: early summer (5,6 months) and autumn (10,11 month), the highest peak in mid May 2010, lasting about 1 months, and a small peak in October; the peak period in 2011 was delayed to half a month, and the HFMD in spring and summer season was slightly higher than that in autumn.
The clinical manifestations were mainly palms, buttocks, plantar macula and oral herpes: 90% (?), more than 60% of the early onset of fever accompanied by a rash, and a few (7.2-11.3%) symptoms of respiratory tract infection. The clinical manifestations of.EV71 infection, such as vomiting, convulsions, startle or headache, were similar to those caused by EV caused by HFMD. In.2010 to 2011, 89 and 144 children were treated with ICU, of which 75 cases (14.9%) and 128 cases (24.9%) had more.EV71 infection than EV infection (P value 0.037 and 0 respectively). All patients with severe hand, foot and mouth disease had enterovirus related encephalopathy, such as viral encephalitis, and respiratory complications. It is mainly neurogenic pulmonary edema.
The partial VP1 region of 33 strains of EV71 (5 in 2009 and 28 in 2010) was amplified and sequenced. By comparison and analysis, the subtype of C4a was determined, and no significant difference was found in the sequence of isolates from Guangdong. Compared with the Fuyang EV71 epidemic strains in 2008, there was no obvious difference, indicating that it was a domestic circulating epidemic strain and the epidemic strain of Taiwan in recent years. Different genetic subtypes showed that the evolutionary origin of the cross straits strains was different. Through Clustal W software, the clinical isolates and the standard strain BrCr (U22521). Taiwan 1998 epidemic strain (Taiwan98) was compared to the VPl region sequence: nucleotide similarity 93.7%-100%, amino acid similarity 97.0%-100%. 22,98 bit amino acid and Taiwan 98 year epidemic strain and standard strain BrCr It's different.
The second part is real-time fluorescence RT-PCR and EV71-IgM capture ELISA method to evaluate the diagnosis of HFMD caused by EV71.
In order to assess the ELISA (?) method for detecting EV71-IgM, we collected 256 sera from EV71 infection from first days to 158 days. It was found that EV71-IgM was detected in first days. The number of cases increased with the number of days increasing and fifth Tianda 100%. After 3 months, the detection rate of serum EV71-IgM was significantly decreased. The acute phase (7 days of onset (?) detection sensitivity) The sensitivity was 90.2% (138/153), and the sensitivity was 93.6% (233/249) within 90 days.
There was a cross reaction with CA16-IgM detection. The experiment showed that 38 EV71-IgM positive in serum of 122 CA16 infection, 14 EV71-IgM positive in 49 Other enterovirus infection sera, 2 sera in 105 other respiratory virus infection sera (from the same respiratory syncytial virus A and B type double infected patients) EV71-IgM positive for other enterovirus, EV The specificity of 71-IgM capture method ELISA was 69.6% (52171.95.95 (?): 65.2-72.4%), for other respiratory virus specific 98.1% (2/105950% confidence interval: 96.5-99.5%), the positive predictive value was 81.3% (95% confidence interval: 76.9-85.1%), negative predictive value 91% (95% confidence interval: 87.5-93.8%).206 EV71 infection serum detected EV71-IgM Positive 199 (95.7%), CA16-IgM positive 58 (28.2%), of which EV71-IgM was higher than CA16-IgM in 56 (96.6%), 119 CA16 infected serum CA16-IgM positive 83 (69.7%), 36 EV71-IgM positive (30.3%), of which CA16-IgM was higher than EV71-IgM (91.7%), the actual infection of the pathogenic IgM detection of higher value, can be correct area Most of the pathogens are infected.
111 cases of anal swabs and serum samples collected on the same day were detected by two methods of ELISA and real time fluorescence RT-PCR. There was no significant difference between two methods to diagnose EV71 infection (McNemar's x 2 test P=0.648), and the consistency was better (Kappa value 0.729).
The third part is the preparation of EV71 virus capsid protein P1 monoclonal antibody.
The full length sequence primers of VP1 ~ VP4 gene were designed according to the EV71 sequence information (FJ194965.1) sequence information. PCR amplification, Kpn I and Hind III double enzyme cut PCR products, connected the carrier pQE30a. The recombinant plasmid was converted into VP1 ~ VP4 protein after the sequencing confirmed that the recombinant plasmid was induced to express the VP1 to VP4 protein, and all were in the form of inclusion body. 8M urea denaturation and gradient refolding were purified by HisBind Resin chromatography column. The purified VP1 ~ 4 protein was purified. The mice were immunized alternately with the recombinant VP4 protein and purified EV71 virus, and the hybridoma cells were cloned through the fusion of murine splenocytes and hybridoma cells. The recombinant egg white VP1 ~ 4 and the virus package were covered with ELISA and indirect immunofluorescence. The target protein was screened by immunoblot.
The recombinant protein VP1, VP2 and VP4, dissolved in 8M urea, were coated with slate, VP2 and VP4, respectively, to detect the clone supernatant of fusion cells, select the clone with high OD value, and further indirect immunofluorescence to respond to EV71 infection and non infected RD cells, and screen the clone of only EV71 infected RD cells. The clone supernatant was purified with the purified EV71 virus. The target protein was identified by the immunoblotting reaction of the protein components separated by the recombinant VP1-VP4 protein by SDS-PAGE glue. 25 monoclonal antibodies against P1 protein were preliminarily obtained: 6 strains of EV71 virus VP1 protein (natural protein), 4 of which were reacted to the recombinant VP1 protein; the 3 strains were only targeted to the recombinant VP2 protein, and the 2 strains against the virus VPl and VP2 eggs. Among them, one strain was targeted against recombinant VP1 and VP2, the other 1 were directed against recombinant protein VP4, and another 13 strains had not identified target proteins.
Summary
Based on the above three parts, the research summaries are as follows:
In 1.2009-2011, the pathogen of hand foot and mouth disease (HFMD) infected with EV was detected in 1.2009-2011. The EV71 infection was 30252 and 221, respectively, 28.1% (30/107), 45.6% (252/553) and 38.7% (221/579), and EV71 was still the main pathogen of hand foot and mouth disease in Guangdong.
In the year of 2.2009-2011, in Guangdong, there were two major onset peaks, namely, early summer (between 5,6 months) and autumn (10,11 month); 1,2 years were the most common age segments, accounted for 85% of the children under 4 years of age, and the age distribution of patients with severe hand foot and mouth disease caused by EV71 infection was similar to this, and there was no statistical difference.
Guangdong EV71 clinical isolates from 3.2009 to 2010 were C4a subtypes, which were not different from those in Guangdong and Fuyang epidemic in Anhui for 08 years. It was a domestic circulating epidemic strain. Compared with the epidemic strains of Taiwan in recent years, it is different gene subtypes, suggesting that the evolutionary origin of the two strains is different, and the standard strain BrCr, and the 1998 epidemic strain of Taiwan, Taiwan. The nucleotide similarity of 93.7%-100% in 98VP1 region is similar to that of amino acid. The amino acid of 22,98 97.0%-100%. is slightly different from that of Taiwan's 98 year old strain and standard strain BrCr.
4. the sensitivity of EV71 induced ELISA EV71-IgM detection in the diagnosis of EV71 induced hand foot and mouth disease was 90.2% (138/153), the specificity was 69.6% (52/171, relative other enterovirus) and 98.1% (2/105, relative to other respiratory viruses), and the positive predictive value was 81.3% (95% confidence interval: 76.9-85.1%) and negative predictive value 91% (95% confidence interval: 87.5-93.8%). There was no significant difference in the diagnosis of EV71 infection with real-time fluorescence RT-PCR (McNemar's chi square test 2), and the consistency was high. The method is suitable for clinical application. Conclusion: there is no significant difference between the two methods (McNemar's chi square test).
5. ELISA EV71-IgM detection was used to detect EV71, although there was a cross reactivity (28.2% and 30.3%) for CA16 infected HFMD sera, but the higher OD value could reflect the type of infected virus.
6. EV71 purified virus and recombinant VP4 protein were used to immunize mice, and 25 monoclonal antibodies against P1 protein were obtained. EV71 virus liquid, VP1-VP4 recombinant protein ELISA, indirect immunofluorescence screening and immunoblotting identification were respectively: 6 strains of VP1 protein, 3 VP2 protein, 1 strains of VP1 and VP22 strain and VP4 protein with clockwise, and 13 strains were not determined.

【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R392;R725.1

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本文編號(hào)：1868626