本文選題：H3亞型禽流感病毒　切入點(diǎn)：分離鑒定　出處：《廣西師范大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：禽流感(Avian influenza, AI)是一種世界范圍性的禽類傳染病,給養(yǎng)禽業(yè)造成巨大的經(jīng)濟(jì)損失,對(duì)人類也存在潛在的威脅。H3亞型禽流感病毒(Avian influenza virus, AIV)為低致病性AIV,不僅可以感染禽類,而且在豬、馬、犬等哺乳動(dòng)物體內(nèi)也可檢測(cè)到。為了解廣西地區(qū)H3亞型AIV的生態(tài)流行趨勢(shì),掌握其遺傳進(jìn)化特點(diǎn)以及與不同宿主(豬、馬、犬和人)的H3亞型流感病毒之間的傳播關(guān)系,同時(shí)建立針對(duì)H3亞型AIV的核酸檢測(cè)技術(shù)來達(dá)到及早診斷和防控的目的,進(jìn)行了如下研究。本研究從2009～2014年活禽市場(chǎng)采集樣品中,通過血清學(xué)試驗(yàn)初步篩選出主要以H3亞型AIV混合感染的毒株,并應(yīng)用雞胚終點(diǎn)稀釋法及血清中和法對(duì)病毒進(jìn)行了分離和純化。為了進(jìn)一步確認(rèn)其基因型,對(duì)分離株HA和NA基因進(jìn)行了克隆及序列測(cè)定,發(fā)現(xiàn)其具有H3N2、H3N6和H3N8三種基因型。對(duì)其中14株分離株進(jìn)行了生物學(xué)特性試驗(yàn),結(jié)果表明這14株分離株均為低致病性禽流感,且均具有優(yōu)先選擇于禽源SA α-2,3-Gal受體結(jié)合的特性,不具有感染人的潛在能力。通過對(duì)14株H3亞型AIV分離株的八個(gè)基因片段(HA、NA、PB2、PB1、PA、NP、NS和M)進(jìn)行克隆、序列測(cè)定和分析,表明14株分離株HA基因裂解位點(diǎn)均只有一個(gè)堿性氨基酸,符合低致病性禽流感的分子特征；與GenBank中不同宿主的H3亞型流感病毒及與分離株的八個(gè)基因片段同源性最高的毒株進(jìn)行各片段核苷酸序列同源性比較,并構(gòu)建各個(gè)基因組分子遺傳進(jìn)化樹。遺傳進(jìn)化分析表明14株分離株各基因組均屬于歐亞譜系的禽源進(jìn)化分支,相鄰年份的分離株各基因組核苷酸同源性較高,進(jìn)化樹分布位置相對(duì)集中,可能來源于相同的祖先；分離年份間隔較大的毒株,各基因組核苷酸序列同源性相對(duì)較低,親緣關(guān)系較遠(yuǎn),具有明顯的時(shí)間差異性,上述結(jié)果表明H3亞型AIV可能處于不斷進(jìn)化中；同一分離株的八個(gè)基因片段來源較為復(fù)雜,可能是由不同亞型AIV發(fā)生基因重排而來；14株分離株與豬源、人源、馬源、犬源H3亞型流感病毒各基因組核苷酸序列同源性較低,親緣關(guān)系較遠(yuǎn),具有明顯的種屬差異性。本研究針對(duì)H3亞型AIV建立了兩種核酸檢測(cè)方法：1)多重RT-PCR方法,包括H3N2亞型AIV雙重RT-PCR和H3N2亞型與M基因三重RT-PCR。2)實(shí)時(shí)熒光定量RT-PCR方法,包括H3N2亞型AIV雙重實(shí)時(shí)熒光定量RT-PCR和H3N8亞型AIV一步法實(shí)時(shí)熒光定量RT-PCR。該方法都實(shí)現(xiàn)了一管多檢的目的,節(jié)省了時(shí)間與成本,同時(shí)也提高了檢測(cè)的準(zhǔn)確性,為臨床樣品的檢測(cè)及H3亞型AIV的防控提供了技術(shù)支撐。
[Abstract]:Avian influenza virus (AII) is a worldwide avian infectious disease, which causes enormous economic losses to the poultry industry and has a potential threat to humans. Avian influenza virus (AIV) is a low pathogenic avian influenza virus, which can not only infect birds. In order to understand the ecological epidemic trend of H3 subtype AIV in Guangxi region, to understand its genetic and evolutionary characteristics and with different hosts (pig, horse, horse), The relationship between the transmission of H3 subtype influenza virus in dogs and humans, and the establishment of nucleic acid detection for H3 subtype AIV for early diagnosis and prevention and control, were studied as follows. This study was carried out from the live poultry market from 2009 to 2014. In order to confirm the genotypes of H3 subtype AIV, the virus was isolated and purified by serological test, and the virus was isolated and purified by chicken embryo end point dilution method and serum neutralization method. The HA and na genes of the isolates were cloned and sequenced. It was found that they had three genotypes of H _ 3N _ 2, H _ 3N _ 6 and H _ 3N _ 8. The biological characteristics of 14 of them were tested. The results showed that the 14 isolates were all low pathogenic avian influenza. All of them were preferentially selected for the binding of SA 偽 -2N 3-Gal receptor, and did not have the potential to infect human beings. Eight gene fragments of 14 H3 subtype AIV isolates were cloned, sequenced and analyzed. The results showed that there was only one basic amino acid in HA gene cleavage site of 14 isolates, which was consistent with the molecular characteristics of low pathogenic avian influenza. The nucleotide sequence homology was compared with H3 subtype influenza virus in different hosts of GenBank and the isolates with the highest homology of eight gene fragments. The genetic evolution analysis showed that all the genomes of 14 isolates belonged to the avian evolutionary branch of Eurasian lineage, and the nucleotide homology of each genome in adjacent years was high. The distribution of the evolutionary tree is relatively concentrated, which may come from the same ancestor. The nucleotide sequence homology of each genome is relatively low, and the genetic relationship is far away. These results suggest that H3 subtype AIV may be in continuous evolution, and that the origin of eight gene fragments of the same isolate is more complex, which may be the result of gene rearrangement from different subtypes of AIV to 14 isolated strains and porcine, human and horse sources. The nucleotide sequence homology of H3 subtype influenza virus in dogs was low and the genetic relationship was far away. In this study, two nucleic acid detection methods for H3 subtype AIV were established. Including H3N2 subtype AIV double RT-PCR and H3N2 subtype and M gene triple RT-PCR.2) real-time fluorescence quantitative RT-PCR method, including H3N2 subtype AIV dual real-time fluorescence quantitative RT-PCR and H3N8 AIV one-step real-time fluorescence quantitative quantitative RT-PCR. It can save time and cost, improve the accuracy of detection, and provide technical support for the detection of clinical samples and the prevention and control of H3 subtype AIV.
【學(xué)位授予單位】：廣西師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S852.65

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本文編號(hào)：1575084