鸭源H9N2亚型AIV分离鉴定与荧光定量RT-PCR检测方法的建立与应用

发布时间：2018-02-01 11:59

本文关键词： H9N2亚型AIV 分离鉴定相对荧光定量PCR 雏鸭排毒病毒载量　出处：《山东农业大学》2017年硕士论文　论文类型：学位论文

【摘要】：H9N2亚型AIV在鸭群中很少引起发病,但家鸭被认为是AIV的巨大储存库,在一定程度上影响着禽流感病毒的发生和传播。目前在我国许多地区的鸭群中分离和检测到H9N2亚型AIV,对于它的潜在危害已经受到越来越多的关注。本研究包括以下三个部分:1.H9亚型AIV的分离鉴定、生物学特性研究以及全基因序列分析本试验从山东济宁送检的疑似H9N2亚型AIV感染的病鸭气管,肺脏等组织中分离得到1株H9亚型禽流感病毒,命名为JN1株。通过血凝血抑、EID50、IVPI、MDT和抗原相关系数等的测定分析和比较JN1株的致病力。结果表明,JN1株的EID50为10-7.54/0.2mL,MDT为72h,IVPI为0.26。JN1株和H9N2标准株抗原相关指数为0.470.5,抗原相关性较差,说明,JN1株已在抗原性上发生改变。根据H9N2亚型AIV在GenBank中的序列,设计引物扩增JN1株的全基因组序列;利用MEGA5.1软件对其8个基因各自绘制系统发育树并进行分析。系统发育分析表明,JN1株的HA与DK/HK/Y280/97在同一分支上,NA、M、NP、PA、PB1、PB2基因与SH/F/98同源性较高,NS基因则与鸭源H5N1亚型AIV NS基因同源性高达99%。HA的裂解位点为PSRSSR↓GL,存在6个潜在糖基化位点,在234位的氨基酸残基为L,NA基因颈部存在缺失。2.H9N2亚型AIV病毒荧光定量PCR快速检测方法的建立与应用根据JN1株的HA基因,在其保守区域设计合成1对特异性引物,同时设计1对扩增内参基因β-actin的引物,扩增片段分别为139 bp,119 bp。以阳性重组质粒作为标准品做标准曲线,建立检测H9N2亚型AIV的相对荧光定量RT-PCR方法。结果表明,HA基因和β-actin基因标准曲线线性关系R值在0.99以上,表现出良好的线性关系;荧光定量PCR最小检出模板浓度约为10 Copies/μL,比RT-PCR灵敏100倍以上;特异性良好;HA基因和β-actin基因的批内、批间变异系数均小于1%。3.H9N2亚型AIV的排毒规律及组织中病毒载量的变化将纯化的H9N2亚型AIV通过点眼滴鼻感染3周龄健康雏鸭,107.84EID50/只。攻毒后,每隔2天采集咽拭子、泄殖腔棉拭子以及血液,并随机剖杀3只。利用建立的检测方法检测病毒的排毒规律及组织中病毒相对表达量,同时测定血清HI抗体效价。结果显示:血清HI试验结果显示,雏鸭接种JN1株之后抗体效价一直维持在较低水平,雏鸭感染H9N2亚型AIV后,第2天即可检测到排毒,在检测过程中,出现了两个排毒高峰,无论是泄殖腔棉拭子还是咽拭子,均在第3天达到排毒高峰,在第11天排毒量降到最低,第13天突然升高,后降低。在排毒过程中,咽拭子排毒量要高于泄殖腔棉拭子。人工感染第2天即可在各个组织中检测到H9N2亚型AIV,感染后7-11天在各组织中的相对表达量较高。H9N2亚型AIV相对表达量最高的器官为气管、肝脏、胰腺和腺胃,相对表达量在0.4~0.6之间,表达量较低的为肺脏、脾脏、肾脏和法氏囊,相对表达量在0.2~0.4之间。本试验分离到的鸭源H9N2亚型AIV,为低毒力株,但对鸭有一定的致病性。利用建立的HA基因的相对荧光定量RT-PCR方法对雏鸭感染H9N2亚型AIV后排毒规律和组织病毒载量测定时发现,在实验室条件下,3周龄雏鸭人工感染H9N2亚型AIV后2-17d均有排毒,2-7d排毒量较多。感染后7-11天组织含毒量较高。
[Abstract]:H9N2 subtype AIV rarely cause disease in ducks, but the duck is considered to be a huge repository of AIV, to a certain extent affect the occurrence and spread of avian influenza virus. At present in many areas of our country the ducks in the separation and detection of H9N2 subtype AIV, the potential harm it has been more attention. This study includes the following three parts: the isolation and identification of 1.H9 subtype AIV, biological characteristics and genome sequence analysis of the test from the suspected disease duck tracheal H9N2 subtype AIV infection in Shandong from Jining, 1 strains of H9 subtype avian influenza virus isolated from lung tissue, named JN1 the blood clotting inhibition. Strains EID50, IVPI, MDT, and the correlation coefficient of determination of antigen analysis and comparison of the pathogenicity of JN1 strain. The results showed that the JN1 strain of EID50 10-7.54/0.2mL, MDT 72h, IVPI 0.26.JN1 strain and the H9N2 strain related antigen index is 0.47 0.5, the antigen poor correlation shows that JN1 strains have been changed. According to the antigenicity of H9N2 subtype AIV in GenBank sequence, amplified genome sequence of JN1 strain primers; phylogenetic tree and analyze the 8 genes each rendering system using MEGA5.1 software. The system development analysis showed that HA and DK/HK/Y280/97 JN1 were in the same branch, NA, M, NP, PA, PB1, PB2 gene and SH/F/98 NS gene homology, with duck origin H5N1 subtype AIV NS gene homologous to the cleavage site of up to 99%.HA for PSRSSR: GL, there are 6 potential glycosylation sites in amino acid residue based on the 234 for the L, the rapid detection method of fluorescence quantitative PCR deletion of.2.H9N2 subtype AIV virus NA gene exists in the neck of the establishment and application of the HA gene of JN1 strain in the conservative region, 1 pairs of primers were designed for amplification of 1, while the design of reference gene beta -actin primers, amplified fragment Some were 139 BP, 119 bp. positive recombinant plasmid as a standard for the standard curve, the relative fluorescence quantitative RT-PCR method was established to detect H9N2 subtype AIV gene. The results showed that the standard curve of HA gene and beta -actin linear relationship between the R value above 0.99, showing a good linear relationship between the fluorescence quantitative PCR minimum detection template; the concentration of Copies/ is about 10 L, 100 times more sensitive than RT-PCR or above; good specificity; HA gene and beta -actin gene in batch, viral shedding pattern and interassay coefficients of variation were less than 1%.3.H9N2 AIV subtype in the quantity change of the purified H9N2 subtype AIV through oral nasal infection 3 weeks old healthy ducklings, only 107.84EID50/. After infection, every 2 days, throat swab, cloacal swabs and blood, and killed randomly 3 rats. Expression of virus excretion and tissue using the detection method of virus detection, measured at the same time Set the HI titers in serum. The results showed that the serum HI test results showed that the ducklings inoculated with JN1 strain after antibody titer remained at a low level, Ducklings Infected with H9N2 subtype AIV after second days can be detected in the detection process, detoxification, detoxification appeared two peak, either the cloacal swab or pharynx swab, reached the peak in the third day detox, detox in eleventh days to a minimum, thirteenth days suddenly increased, and then decreased. In the process of detoxification, detoxification of throat swab was higher than the cloacal swab. Artificial infection second days in all tissues detected by H9N2 subtype AIV infection 7-11 days in different tissues relative higher expression of.H9N2 subtype AIV relative expression of the highest organ of the trachea, liver, pancreas and stomach, the relative expression between 0.4~0.6, the low expression level in lung, spleen, kidney and bursa, relative expression in 0.2~0.4 . in the test was duck origin H9N2 subtype AIV, but for the low virulent strains, there is a certain pathogenicity of duck. The relative fluorescence quantitative RT-PCR method for HA gene based discovery of excretion of virus load test and tissue Ducklings Infected with H9N2 subtype AIV, under the condition of laboratory, 3 week old Ducklings Infected with H9N2 subtype AIV 2-17d were 2-7d after detoxification, detoxification was more higher. 7-11 days after infection of virus content in tissue.

【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.65

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