流感病毒裂解疫苗中外源性禽白血病病毒荧光定量PCR检测方法的研究

发布时间：2018-06-30 11:18

本文选题：禽白血病病毒 + 外源性　；参考：《北京理工大学》2015年硕士论文

【摘要】：流感病毒裂解疫苗作为预防大规模流感疫情的重要预防手段,能有效的预防流感引起的疾病和死亡。接种流感疫苗是被公认的预防流感发生与传播的最佳方法,用鸡胚作为病毒生长介质也是目前流感疫苗生产最常用的培养基质。在2010版《中国药典》新增流感病毒裂解疫苗品种,其中规定明确要求对其主种子批中的外源性禽白血病病毒(Avian Leucosis Virus,ALV)进行检测,检测结果应为阴性。禽白血病病毒的检测目前主要依赖于病毒分离培养及酶联免疫吸附。血清学诊断由于检测时间长,需制备特异性抗体,存在生物安全隐患等不足之处。随着分子生物学检测技术的发展,为禽白血病病毒的准确检定提供了可能。应用普通PCR和RT-PCR方法可在1天内进行禽白血病病毒的鉴定和分型诊断,但两种方法均易造成标本间的交叉污染而得到假阳性结果。Taqman实时荧光定量PCR技术,相对于常规的PCR技术而言,具有更高的敏感性、特异性。而且还具有快速、高通量、污染少等优点。从GenBank下载ALV-A、ALV-B、ALV-C、ALV-J等病毒基因组序列,寻找位于LTR区域的保守序列,设计引物探针。扩增保守区,获得质粒标准品,将含有保守序列的质粒标准品经过10倍系列稀释后,进行荧光定量PCR得到相应的扩增曲线和标准曲线。得到的标准曲线相关系数R2=0.998,扩增效率Eff=96.5%所做的标准曲线的有较好的线性关系。倍比稀释质粒标准品,从1×109稀释到1×101copies/μL。考察其敏感性,敏感性可达到10-1copies/μL。用建立的荧光定量PCR方法对ALV-A、ALV-B、ALV-C、ALV-J等标准质粒进行检测,均可出现“S”型扩增曲线。说明该方法可检测四种亚型的禽白血病病毒。用建立的荧光定量PCR方法对小鼠白血病病毒、H1N1型流感病毒、H7N9型流感病毒等进行检测,扩增结果皆为阴性。表明所建立的针对外源性禽白血病病毒的荧光定量PCR方法特异性强,与其他病毒无交叉反应。本研究成功设计了针对外源性禽白血病病毒的特异性引物和探针,可实现对流感病毒裂解疫苗主种子批中的禽白血病病毒的快速检定,具有快速、敏感、特异等特点,并可对样品中病毒进行定量。
[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.
【学位授予单位】：北京理工大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.65;R373

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