發(fā)布時間：2018-06-30 11:18

  本文選題：禽白血病病毒 + 外源性��； 參考：《北京理工大學(xué)》2015年碩士論文

【摘要】：流感病毒裂解疫苗作為預(yù)防大規(guī)模流感疫情的重要預(yù)防手段,能有效的預(yù)防流感引起的疾病和死亡。接種流感疫苗是被公認(rèn)的預(yù)防流感發(fā)生與傳播的最佳方法,用雞胚作為病毒生長介質(zhì)也是目前流感疫苗生產(chǎn)最常用的培養(yǎng)基質(zhì)。在2010版《中國藥典》新增流感病毒裂解疫苗品種,其中規(guī)定明確要求對其主種子批中的外源性禽白血病病毒(Avian Leucosis Virus,ALV)進(jìn)行檢測,檢測結(jié)果應(yīng)為陰性。禽白血病病毒的檢測目前主要依賴于病毒分離培養(yǎng)及酶聯(lián)免疫吸附。血清學(xué)診斷由于檢測時間長,需制備特異性抗體,存在生物安全隱患等不足之處。隨著分子生物學(xué)檢測技術(shù)的發(fā)展,為禽白血病病毒的準(zhǔn)確檢定提供了可能。應(yīng)用普通PCR和RT-PCR方法可在1天內(nèi)進(jìn)行禽白血病病毒的鑒定和分型診斷,但兩種方法均易造成標(biāo)本間的交叉污染而得到假陽性結(jié)果。Taqman實(shí)時熒光定量PCR技術(shù),相對于常規(guī)的PCR技術(shù)而言,具有更高的敏感性、特異性。而且還具有快速、高通量、污染少等優(yōu)點(diǎn)。從GenBank下載ALV-A、ALV-B、ALV-C、ALV-J等病毒基因組序列,尋找位于LTR區(qū)域的保守序列,設(shè)計(jì)引物探針。擴(kuò)增保守區(qū),獲得質(zhì)粒標(biāo)準(zhǔn)品,將含有保守序列的質(zhì)粒標(biāo)準(zhǔn)品經(jīng)過10倍系列稀釋后,進(jìn)行熒光定量PCR得到相應(yīng)的擴(kuò)增曲線和標(biāo)準(zhǔn)曲線。得到的標(biāo)準(zhǔn)曲線相關(guān)系數(shù)R2=0.998,擴(kuò)增效率Eff=96.5%所做的標(biāo)準(zhǔn)曲線的有較好的線性關(guān)系。倍比稀釋質(zhì)粒標(biāo)準(zhǔn)品,從1×109稀釋到1×101copies/μL�？疾炱涿舾行�,敏感性可達(dá)到10-1copies/μL。用建立的熒光定量PCR方法對ALV-A、ALV-B、ALV-C、ALV-J等標(biāo)準(zhǔn)質(zhì)粒進(jìn)行檢測,均可出現(xiàn)“S”型擴(kuò)增曲線。說明該方法可檢測四種亞型的禽白血病病毒。用建立的熒光定量PCR方法對小鼠白血病病毒、H1N1型流感病毒、H7N9型流感病毒等進(jìn)行檢測,擴(kuò)增結(jié)果皆為陰性。表明所建立的針對外源性禽白血病病毒的熒光定量PCR方法特異性強(qiáng),與其他病毒無交叉反應(yīng)。本研究成功設(shè)計(jì)了針對外源性禽白血病病毒的特異性引物和探針,可實(shí)現(xiàn)對流感病毒裂解疫苗主種子批中的禽白血病病毒的快速檢定,具有快速、敏感、特異等特點(diǎn),并可對樣品中病毒進(jìn)行定量。
[Abstract]:Influenza virus lytic vaccine, as an important preventive means to prevent large-scale influenza epidemic, can effectively prevent illness and death caused by influenza. Inoculation with influenza vaccine is the best method to prevent the occurrence and transmission of influenza. Chicken embryo is also the most commonly used culture medium for influenza vaccine production. In the 2010 edition of the Chinese Pharmacopoeia, new influenza virus lytic vaccine varieties were added. It was stipulated that Avian Leucosis virus (Avian Leukosis virus) should be detected in its main seed batches, and the results should be negative. The detection of avian leukemia virus mainly depends on virus isolation, culture and enzyme linked immunosorbent assay (Elisa). Because of the long detection time, the serological diagnosis needs to prepare specific antibodies, which has some disadvantages such as potential biosafety and so on. With the development of molecular biological detection technology, it is possible for the accurate detection of avian leukemia virus. Ordinary PCR and RT-PCR can be used to identify and type avian leukemia virus within one day. However, both methods are easy to cause cross-contamination between specimens and obtain false positive results. Taqman real-time quantitative PCR technique is used. Compared with the conventional PCR technique, it has higher sensitivity and specificity. And also has the advantages of fast, high throughput, less pollution and so on. The genome sequences of ALV-Agna ALV-BUE ALV-CnALV-J and other viruses were downloaded from GenBank to search for the conserved sequences located in the LTR region, and primer probes were designed. The conserved region was amplified and plasmid standard was obtained. The corresponding amplification curve and standard curve were obtained by fluorescence quantitative PCR after diluting the standard plasmid containing the conserved sequence. The correlation coefficient of the standard curve R _ (2) is 0.998 and the amplification efficiency is 96.5%. There is a good linear relationship between the standard curve and the standard curve. The standard plasmid was diluted from 1 脳 10 ~ 9 to 1 脳 101copies/ 渭 L. The sensitivity can reach 10-1copies/ 渭 L. The standard plasmids such as ALV-AV B, ALV-C, ALV-J and so on were detected by the established fluorescent quantitative PCR method, and the "S" type amplification curve could be found in these plasmids. This method can be used to detect four subtypes of avian leukemia virus. The FQ-PCR method was used to detect the H7N9 influenza virus of murine leukemia virus, and the results were negative. The results showed that the fluorescent quantitative PCR method for exogenous avian leukemia virus was specific and had no cross reaction with other viruses. In this study, specific primers and probes for exogenous avian leukemia virus were designed successfully, which can be used for rapid detection of avian leukemia virus in the main seed batches of influenza virus lytic vaccine. It has the characteristics of fast, sensitive, specific and so on. The virus in the sample can be quantified.
【學(xué)位授予單位】：北京理工大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S852.65;R373

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