本文選題：多重?zé)晒舛縍T-PCR + 甲型流感H3N2　； 參考：《浙江大學(xué)》2015年碩士論文

【摘要】：目的：建立一種快速、敏感、特異的五重?zé)晒舛縍T-PCR方法檢測甲型流感的不同亞型,并對2010～2013年杭州地區(qū)甲型流感H3N2進(jìn)行分子流行病學(xué)調(diào)查,通過選擇壓力分析為疫苗研制提供依據(jù)。方法：1.設(shè)計(jì)甲型禽流感病毒基質(zhì)蛋白(M)區(qū)、H3、H5、H7及內(nèi)參基因(RNaseP,RP)的引物及Taqman探針,并對RT-PCR反應(yīng)體系中,五套引物及探針的濃度進(jìn)行優(yōu)化。2.合成各基因標(biāo)準(zhǔn)品片段,將其連接到質(zhì)粒載體PmdTM19-T Simple Vector上進(jìn)行轉(zhuǎn)化和培養(yǎng)。經(jīng)鑒定后提取質(zhì)粒DNA,利用NanoDrop ND-2000核酸檢測儀測量質(zhì)粒DNA的濃度,確定DNA的拷貝數(shù)作為敏感度的標(biāo)準(zhǔn)品定量母液。根據(jù)實(shí)驗(yàn)需要,將標(biāo)準(zhǔn)品定量母液稀釋至所需最高濃度(107copies/mL),并連續(xù)10倍稀釋至最低濃度(102copies/mL),然后用本方法和WHO推薦的方法進(jìn)行靈敏度比較。3.在本方法的反應(yīng)體系中加入其它18種呼吸道病原微生物[乙型流感病毒,禽流感H5N3、H5N1、H9N2病毒,人副流感病毒Ⅰ～Ⅲ型,呼吸道合胞病毒A和B型,肺炎支原體,銅綠假單胞菌,肺炎克雷伯菌,鮑曼不動(dòng)桿菌,金黃色葡萄球菌,白念珠菌]提取的模板,檢測反應(yīng)體系的特異性。4.將確定好的各質(zhì)粒DNA濃度,根據(jù)實(shí)驗(yàn)需要,稀釋至所需要的濃度(106copies/mL),連續(xù)三次10倍稀釋至(104copies/mL),用本方法分別檢測三次,檢測反應(yīng)體系的(批內(nèi))重復(fù)性,并與WHO推薦方法的結(jié)果進(jìn)行比較。5.用本方法對262例疑似患者與正常入的痰液標(biāo)本進(jìn)行檢測,并與上海之江生物有限公司生產(chǎn)的市售試劑盒的方法結(jié)果比較。6.2010年1月至2013年12月浙江大學(xué)附屬第一醫(yī)院流感監(jiān)測網(wǎng)絡(luò)相關(guān)數(shù)據(jù),各年度流感樣病例分別為：2406例、995例、766例和1156例。分別采用研究試劑所有病例的咽拭子均進(jìn)行H3N2流感病毒檢測,并經(jīng)過血凝抑制實(shí)驗(yàn)和流感病毒核酸監(jiān)測證實(shí)。7.病原學(xué)檢測：樣本先進(jìn)行流感病毒核酸檢測,采用五重RT-PCR檢測試劑盒。按照不同年份、性別以及年齡段對所有結(jié)果進(jìn)行統(tǒng)計(jì)分析。8.序列收集：所有H3N2的HA (hemagglutinin)與NA (neuraminidase)基因完整序列均在NCBI上搜索查找獲取,搜索關(guān)鍵詞分別為:"China", "Hangzhou", "Hong Kong"加"H3N2 HA gene complete cds","H3N2 NA gene complete cds",香港地區(qū)H3N2基因序列并入中國整個(gè)國家序列中。9.HA與NA基因序列篩選與比對：依據(jù)病毒的分離地、時(shí)間、宿主為前提條件進(jìn)行整合,條件相同序列只取一條。H3N2基因序列按照地區(qū)分為中國HA(133)、杭州HA(69)和杭州NA(69)三組。分組的序列分別用軟件Clustal X(v1.8)進(jìn)行比對、分析、剪切對齊。杭州地區(qū)的序列比對完成后,直接運(yùn)用Mega(v6.0)軟件,采用鄰位相接法進(jìn)行系統(tǒng)進(jìn)化樹分析。用FigTree軟件對圖形進(jìn)行編輯。10.分子鐘與人口感染動(dòng)力學(xué)分析：利用jModeltest軟件找出序列最適堿基替換模型,按照手冊中要求參數(shù)設(shè)置(http://code.google.com/p/jmodeltest2).再使用Beast軟件中貝葉斯MCMC (Bayesian Markov chain Monte Carlo)法對數(shù)據(jù)集進(jìn)行堿基替換速率、最近共同祖先(Time of most recent common ancestor, TMRCA)和Skyline plot分析,依據(jù)操作手冊中要求的參數(shù)(http://beast.bio.ed.ac.uk/Tutorials/)進(jìn)行設(shè)置,我們建立一個(gè)嚴(yán)格分子鐘的指數(shù)生長模型,通過Tracer軟件對所得結(jié)果進(jìn)行分析,并規(guī)定有效樣本大小(Effective sampling Size, ESS)200,最終計(jì)算分子鐘(堿基替換速率)與時(shí)間人口感染動(dòng)力學(xué)關(guān)系圖,用Beast軟件包中TreeAnnotator程序,計(jì)算獲最大可信樹(MCCT, Maximum clade credibility tree),用FigTree對所得到的樹進(jìn)行編輯,獲得H3N2病毒的HA基因在時(shí)間標(biāo)尺與地理位置上的進(jìn)化圖。11.甲型流感病毒H3N2選擇壓力分析：選擇壓力(dN-dS)可以反映病毒在收到外界環(huán)境進(jìn)行選擇時(shí)的進(jìn)化情況,其中,dN (non-synonymous substitutions)代表非同義突變率,dS (synonymous substitutions)表示同義突變率。選擇壓力計(jì)算時(shí)以密碼子發(fā)生非同義突變率dN與同義突變率dS的差值來確定選擇的方向性,中性選擇(dN=dS,dN-dS=O),正向選擇(dN-dS0),凈化選擇(負(fù)向選擇,dN-dS0).整組基因選擇壓力計(jì)算使用在線的Datamonkey webserve計(jì)算,導(dǎo)向進(jìn)化樹使用Neighbor Joining方法,選用在線檢測的推薦模型作為序列的進(jìn)化模型,因其序列數(shù)50,算法采用singlelikelihood ancestor counting (SLAC),使用Datamonkey在線網(wǎng)站提供的序列選擇壓力分析程序進(jìn)行選擇壓力分析。12.與選擇壓力(正向選擇)相關(guān)的蛋白質(zhì)表達(dá)：在PDB數(shù)據(jù)庫中找出與H3N2基因密碼子相似程度較高(30%)的蛋白質(zhì)結(jié)構(gòu),以此蛋白質(zhì)結(jié)構(gòu)進(jìn)行同源建模,比較各選擇壓力位點(diǎn)下的蛋白質(zhì)結(jié)構(gòu)的變化。分別在蛋白質(zhì)空間三維結(jié)構(gòu)圖中進(jìn)行標(biāo)記。結(jié)果：1.引物與探針的設(shè)計(jì)與濃度優(yōu)化：從美國NCBI基因庫下載涵蓋國內(nèi)外FluA及H3、H5、H7亞型的多條基因序列,用DNAman軟件對其進(jìn)行同源性比較,確定以上病毒基因組的保守區(qū),用Primer Express3.0軟件在其保守區(qū)設(shè)計(jì)高度特異性的引物與TaqMan探針,并進(jìn)行BLAST序列比對驗(yàn)證引物特異性。引物與探針濃度采用矩陣法進(jìn)行,以獲得的Ct值較小而熒光強(qiáng)度最大的濃度為反應(yīng)體系最合適濃度。2.五重?zé)晒舛縍T-PCR反應(yīng)體系的靈敏度：五重?zé)晒舛縍T-PCR反應(yīng)在檢測甲型禽流感病毒基質(zhì)蛋白(M)區(qū)、H3、 H5、 H7及RNase P的合成片段時(shí),各自靈敏度與WHO推薦的方法一致,均達(dá)到了在102copies/mL時(shí)能擴(kuò)增出條帶。3.五重?zé)晒舛縍T-PCR反應(yīng)體系的特異性：將上述18種呼吸道病原菌的核酸提取物作為模板分別加入多重?zé)晒舛縍T-PCR進(jìn)行測定,除流感病毒出現(xiàn)很好的陽性結(jié)果外,其它所有病毒的檢測結(jié)果均呈陰性。特異性達(dá)到了100%。4.五重?zé)晒舛縍T-PCR反應(yīng)體系的重復(fù)性(批內(nèi))：不同濃度核酸各自的檢測Ct值標(biāo)準(zhǔn)差在0.193～0.451之間,變異系數(shù)(CV)最高達(dá)1.999%,本方法具有較好的批內(nèi)重復(fù)性。5.五重?zé)晒舛縍T-PCR反應(yīng)體系的檢測符合率：使用本方法與已有試劑對臨床262例疑似患者和正常人的痰液標(biāo)本應(yīng)用多重?zé)晒舛縍T-PCR方法進(jìn)行檢測,共檢查出甲型流感病毒52例,其中H3N2型11例,H7N9型4例,H5N1未檢測到陽性病例,與之江生物有限公司的市售試劑盒檢測結(jié)果的符合度達(dá)100%。6.2010～2013年杭州地區(qū)H3N2感染率分析,不同年度間H3N2感染率差異具有統(tǒng)計(jì)學(xué)意義(χ2=14.004,P0.05),不同性別和年齡組間差異無統(tǒng)計(jì)學(xué)意義(χ2=3.552,χ2=2.691,P0.05)。杭州地區(qū)2010年與2012,2013年甲型流感H3N2的HA、NA序列進(jìn)化來自廣東省甲型流感H3N2進(jìn)化的不同時(shí)間點(diǎn)的兩個(gè)分支。7.我國流行甲型流感病毒H3N2的HA基因堿基替換速率為1.6584×10-3(95%可信區(qū)間：1.5325×10-3～1.7988×10-3), TMRCA:1945年(95%可信區(qū)間：1940～1952年),人口感染規(guī)模評價(jià)出現(xiàn)了兩次高峰且感染率總趨勢呈現(xiàn)上升狀態(tài)。8.通過SLA法計(jì)算可得到陽性選擇位點(diǎn)4個(gè),14,153,209,278。陰性位點(diǎn)有238個(gè)。結(jié)論：1.多重?zé)晒舛縍T-PCR技術(shù)是利用TaqMan技術(shù)在普通PCR原有的一對特異性引物基礎(chǔ)上增加了一條特異性的熒光雙標(biāo)記探針。利用該探針能與病原核酸特異性結(jié)合,而且結(jié)合部位位于引物結(jié)合區(qū)域,探針的5'端和3'端分別標(biāo)記不同的熒光素,在PCR擴(kuò)增過程中,利用檢測系統(tǒng)中熒光量的變化,通過檢測儀檢測并記錄熒光信號的變化判定檢測結(jié)果,我們利用此原理建立了多重?zé)晒釸T-PCR法檢測不同的流感亞型。2.檢測方法的評價(jià)：通過標(biāo)準(zhǔn)毒株與臨床樣本的檢測比較,發(fā)現(xiàn)我們建立的多重?zé)晒釸T-PCR特異性和靈敏度均達(dá)到了WHO推薦方法的特異性和靈敏度水平,而且我們的方法更快速,簡便,并只需一次反應(yīng)可同時(shí)檢測甲型禽流感病毒和H3、H5、H7不同甲型禽流感不同亞型。3.臨床標(biāo)本驗(yàn)證：通過分別使用我們試劑與之江在售試劑,同時(shí)檢測臨床病人與正常人標(biāo)本后,我們得出兩者符合率達(dá)到了100%。4.通過對該方法的多角度評價(jià),可知該方法具有快速、穩(wěn)定、靈敏度和特異性高、重復(fù)性好的特點(diǎn),對于臨床上檢測流感病毒及其不同亞型具有一定的意義。5.我們推測2012年后的H3N2病毒株可能是其他省份病毒遷徙所致；2013年病毒株HA與NA基因序列,均處于2012年病毒株樹分支的下級,我們推斷2013年的病毒株為2012年病毒株復(fù)蘇后感染再傳播,2012年H3N2病毒株感染后,使部分感染人群產(chǎn)生獲得免疫,對于此病毒株再次感染具有一定的保護(hù)作用,致使2013年杭州地區(qū)H3N2病毒流感樣人群中感染率下降。6.H3N2基因堿基置換速率為1.6525×10-3(95%可信區(qū)間：1.4796×10-3～1.8287×10-3),要明顯低于H1N1(7.06×10-3,95%可信區(qū)間：5.4×10-3-8.8×10-3)、H5N1(8.87×10-3,95%可信區(qū)間：7.0×10-3～10.72×10-3)等高致病性禽流感的堿基置換速率,這也是H3N2在我國流行的感染病毒株,多以低變異株為主的主要原因。由堿基置換速率我們推測H3N2病毒H3基因的產(chǎn)生時(shí)間：1945年(95%可信區(qū)間：1940～1952年),由于H3N2病毒存在一組未在人類感染的病毒進(jìn)化分支,因此剔除未感染人病毒組,計(jì)算人感染H3N2的H3基因產(chǎn)生時(shí)間為：1960年(95%可信區(qū)間：1957～1962年)與實(shí)際H3N2流行發(fā)生時(shí)間相符。同時(shí)H3N2病毒在我國感染的規(guī)模與時(shí)間的分布出現(xiàn)了兩次高峰且感染率總趨勢呈現(xiàn)上升狀態(tài)。7.可將HA1基因的153(137)位于A抗原區(qū),209(193)位于B抗原區(qū),278(262)位于E抗原區(qū),作為研制H3N2流感疫苗的依據(jù),在H3N2流行中應(yīng)重點(diǎn)觀察該病毒在這些區(qū)域的變異情況
[Abstract]:Objective: to establish a fast, sensitive and specific five heavy fluorescence quantitative RT-PCR method to detect the different subtypes of influenza A, and to investigate the molecular epidemiology of influenza A influenza A (H3N2) in Hangzhou for 2010~2013 years, and to provide the basis for the development of the vaccine by selecting pressure analysis. Method: 1. to design the matrix protein (M) region of avian influenza A virus (H3). H5, H7 and RNaseP, RP (RNaseP, RP) primers and Taqman probes, and the concentration of five sets of primers and probes in the RT-PCR reaction system were optimized for.2. synthesis of each gene standard fragment, which was connected to the plasmid vector PmdTM19-T Simple Vector to be transformed and cultured. The plasmid DNA was extracted and detected by NanoDrop nucleic acid. The concentration of plasmid DNA was measured by the instrument, and the copy number of DNA was determined as the standard quantitative maternal liquid for sensitivity. According to the experimental needs, the standard quantitative mother liquid was diluted to the maximum required concentration (107copies/mL), and 10 times diluted to the lowest concentration (102copies/mL), and then the sensitivity was compared with this method and the method recommended by WHO to.3. in this method. In the reaction system, 18 other respiratory pathogenic microorganisms (influenza B virus, avian influenza H5N3, H5N1, H9N2 virus, human parainfluenza virus I to III, respiratory syncytial virus A and B, Mycoplasma pneumoniae, Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter Bauman, Staphylococcus aureus, Candida albicans) were extracted. The specific.4. of the reaction system will determine the good plasmid DNA concentration, dilute it to the required concentration (106copies/mL) according to the experimental needs, dilute it to (104copies/mL) three times continuously, and detect three times by this method, and detect the repeatability of the reaction system (in batch), and compare with the results of the WHO recommendation method,.5. with this method to 262. Cases of suspected patients and normal sputum specimens were tested, and compared with the method of marketing reagents produced by Shanghai Zhijiang biological Co., Ltd., the data of influenza surveillance network in the First Affiliated Hospital of Zhejiang University from January to December 2013.6.2010 were compared. The annual influenza like cases were 2406 cases, 995 cases, 766 cases and 1156 cases respectively. Do not use the swabs of all the cases of the reagents to detect the H3N2 influenza virus, and through the hemagglutination inhibition test and the influenza virus nucleic acid monitoring to confirm the.7. pathogenic test: the sample first carried out the detection of influenza virus nucleic acid, and the five RT-PCR detection kit was used. All the results were counted according to the different years, sex and age. .8. sequence collection: all H3N2 HA (hemagglutinin) and NA (neuraminidase) gene complete sequences are searched and obtained on NCBI, and the search key words are "China", "Hangzhou", "Hong Kong" and "Hong Kong". The sequence screening and comparison of.9.HA and NA gene sequences in the column: Based on the isolation, time and host of the virus, the same sequence only takes one.H3N2 sequence according to the region of China HA (133), Hangzhou HA (69) and Hangzhou NA (69). The sequence of the group is compared with the software Clustal X (v1.8), analysis, shear pairs, respectively. After the sequence alignment of Hangzhou area is completed, the Mega (V6.0) software is used directly to analyze the phylogenetic tree with the neighbor connection method. The graph is used to edit the.10. molecular clock and the dynamics analysis of the population infection: using the jModeltest software to find the optimal base replacement model of the sequence, set the parameters in the manual (HTTP). //code.google.com/p/jmodeltest2). Then use the Bayesian MCMC (Bayesian Markov chain Monte Carlo) method in the Beast software to carry out the base substitution rate for the data set. Als/) set up, we set up an exponential growth model of a strict molecular clock, analyze the results through Tracer software, and specify the size of the effective sample (Effective sampling Size, ESS) 200, and finally calculate the relationship diagram of the molecular clock (base substitution rate) and the temporal human mouth infection, and use the TreeAnnotator process in the Beast software package. In order, the MCCT, Maximum clade credibility tree was obtained, and the tree was edited with FigTree to obtain the evolution diagram of the HA gene of the H3N2 virus in the time scale and geographic location. The pressure analysis of the H3N2 selection of influenza A virus (.11.) of the influenza A virus was analyzed. The selection of pressure (dN-dS) could reflect the selection of the virus in the environment of receiving the environment. DN (non-synonymous substitutions) represents the non synonymous mutation rate, and dS (synonymous substitutions) represents the synonymous mutation rate. The selection pressure is calculated with the difference between the non synonymous mutation rate dN and the synonymous mutation rate dS in the selection pressure calculation, and the neutral selection (dN=dS, dN-dS=O), positive selection (dN-dS0). The purification selection (negative selection, dN-dS0). The whole group of gene selection pressure calculation uses the online Datamonkey webserve calculation, directing the evolutionary tree using the Neighbor Joining method, selecting the recommended model for on-line detection as the evolutionary model of the sequence, because the sequence number is 50, the algorithm uses singlelikelihood ancestor counting (SLAC), and uses Datamonkey. The sequence selection pressure analysis program provided by the online web site selects the protein expression associated with the selection pressure (positive selection) of the pressure analysis.12.: in the PDB database, the protein structure with a higher similarity to the H3N2 gene codon (30%) is found, and the protein structure is used for homologous modeling, and the eggs under the selection of pressure loci are compared. The changes in white matter structure are marked in the three-dimensional structure map of protein. Results: design and concentration optimization of 1. primers and probes: download the multiple gene sequences of FluA and H3, H5, H7 subtypes at home and abroad from the American NCBI gene pool, and compare the homology with DNAman software to determine the conserved area of the above virus genome. Primer Express3.0 software was used to design highly specific primers and TaqMan probes in the conserved area, and the specificity of primers was verified by BLAST sequence alignment. The primer and probe concentration was carried out by matrix method to obtain the lowest Ct value and the maximum concentration of fluorescence intensity was.2. five heavy fluorescence quantitative RT-PCR reactant. Sensitivity: the sensitivity of five heavy fluorescence quantitative RT-PCR reaction in detecting the matrix protein (M) region of avian influenza A (M), H3, H5, H7 and RNase P, each sensitivity is consistent with the recommended method of WHO, all of which are able to amplify the specificity of the band.3. five heavy fluorescein quantitative RT-PCR reaction system at 102copies/mL: the above 18 kinds of calls are called. The nucleic acid extracts of the pathogen of the pathogeny were tested with multiple fluorescent quantitative RT-PCR as the template. Except for the positive results of the influenza virus, all the other viruses were negative. The specificity reached the repeatability of the 100%.4. five heavy fluorescence quantitative RT-PCR reaction system (in batch): different concentrations of nucleic acids respectively The standard deviation of Ct values is between 0.193 and 0.451, and the coefficient of variation (CV) is up to 1.999%. This method has a good detection coincidence rate of the internal repetitive.5. five heavy fluorescence quantitative RT-PCR reaction system: using this method and the existing reagents, the multiple fluorescence quantitative RT-PCR method is applied to the sputum specimens of 262 clinically suspected patients and normal people. A total of 52 cases of influenza A virus were examined, including 11 cases of H3N2, 4 cases of type H7N9, and no positive cases detected by H5N1. The coincidence of the test results with the marketing reagent box of Zhijiang biological Co., Ltd. reached 100%.6.2010 ~ 2013 in Hangzhou area, and the difference of H3N2 infection rate between different years was statistically significant (x 2=14.004, P) 0.05) there was no statistical difference between different sex and age groups (x 2=3.552, X 2=2.691, P0.05). The HA of influenza a H3N2 in 2010 and 20122013 years in Hangzhou region, NA sequence evolution came from two branches of the different time points of the evolution of influenza A influenza A in Guangdong Province,.7., the rate of the HA gene base substitution of influenza A influenza A virus in China was 1.658 4 x 10-3 (95% confidence interval: 1.5325 x 10-3 ~ 1.7988 x 10-3), TMRCA:1945 years (95% confidence interval: 1940~1952 years), population infection scale evaluation appeared in two peaks and the total trend of infection rate showed an increasing state..8. can be calculated by SLA method by SLA method and 14153209278. negative loci have 238. The optical quantitative RT-PCR technique is a specific fluorescent double labeled probe based on TaqMan technology on the basis of a pair of specific primers in common PCR. Using the probe, the probe can combine with the specific nucleic acid of the pathogen, and the binding site is located in the primer binding region, and the 5'and 3' terminals of the probe are labeled with different fluorescein, and the amplification is amplified in PCR. In the process, we use the detector to detect and record the change of fluorescence signal by detecting and recording the changes of fluorescence signal in the detection system. By using this principle, we establish a multiple fluorescence RT-PCR method for detecting different influenza subtypes.2. detection methods. The specificity and sensitivity of the heavy fluorescence RT-PCR have reached the specificity and sensitivity of the WHO recommendation method, and our method is more rapid and simple, and only one response can be used to detect the avian influenza A virus and H3, H5, H7 different subtypes of avian influenza a different subtype.3.. After selling the reagents and testing the clinical and normal human specimens, we found that the coincidence rate reached 100%.4. through the multi angle evaluation of the method. It is known that the method has the characteristics of rapid, stable, high sensitivity, high specificity and good repeatability. It has a certain significance for the detection of influenza virus and its different subtypes in clinical.5.. It is presumed that the H3N2 virus strain after 2012 may be the result of the virus migration in other provinces, and the sequence of HA and NA in 2013 is in the lower grade of the branch of the virus tree in 2012. We infer that the virus strain of 2013 is transmitted again after the 2012 virus strain is resuscitated, and after the infection of the H3N2 virus in 2012, some infected people will be immune. It has a certain protective effect on the reinfection of the virus strain, resulting in the decrease of.6.H3N2 gene base replacement rate of 1.6525 H3N2 10-3 (95% confidence interval: 1.4796 x 10-3 to 1.8287 x 10-3) in 2013, which is significantly lower than that of H1N1 (7.06 x 10-3,95% confidence interval: 5.4 x 10-3-8.8 * 10-3), H5N1 (8.87). X 10-3,95% confidence interval: 7 * 10-3 ~ 10.72 * 10-3) base replacement rate of high pathogenic avian influenza, which is the main reason that H3N2 is prevalent in our country, mainly with low variation strain. By the base replacement rate, we speculate the generation time of the H3N2 virus H3 gene: 1945 (95% confidence interval: 1940~1952 years), because The H3N2 virus has a group of uninfected virus evolution branches. Therefore, the H3 gene generation time of the human infected H3N2 is: 1960 (95% confidence interval: 1957~1962 years) is in accordance with the actual incidence of the actual H3N2 epidemic. Meanwhile, the distribution of the H3N2 virus in the scale and time of the infection in our country is two times higher The general trend of peak and infection rate showed a rising state of.7., which could locate 153 (137) of HA1 gene in A antigen region, 209 (193) located in B antigen region, 278 (262) in E antigen region, as the basis for developing H3N2 influenza vaccine. In H3N2 epidemic, the variation of the virus in these regions should be observed.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R446.5

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1 黃維娟;成艷輝;李希妍;趙翔;郭俊峰;王釗;譚敏菊;李明;隋z裚";隗合江;陳瑤瑤;肖寧;藍(lán)雨;王大燕;舒躍龍;;2011～2012年度中國H3N2亞型流感病毒病原學(xué)特征分析[J];病毒學(xué)報(bào);2013年03期

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