猪细小病毒论文：猪细小病毒、伪狂犬病病毒和猪圆环病毒2型多重pcr检测方法的建立及应用

猪细小病毒论文：猪细小病毒、伪狂犬病病毒和猪圆环病猪细小病毒论文：猪细小病毒、伪狂犬病病毒和猪圆环病 毒毒 2 2 型多重型多重 PCRPCR 检测方法的建立及应用检测方法的建立及应用 【中文摘要】猪细小病毒(Porcine parvovirus,PPV)、猪伪狂 犬病病毒(Pseudorabies virus,PRV)、猪圆环病毒 2 型(Porcine circovirus type 2,PCV-2)是当前影响养猪业发展的 3 种重要的病 原。由于 PPV、PRV、PCV-2 单独感染或混合感染均能引起猪发病,造 成母猪的繁殖障碍或(和)感染猪的免疫抑制,为其他病原的继发感染 提供了便利,甚至会造成猪群中疫病的流行,使养殖效益蒙受损失。 聚合酶链反应(polymerase chain reaction, PCR)具有快速、敏感、 特异等优点,自发明以来在病原的特异性检测方面表现出明显的优势,在 动物疫病诊断和病原检测上也得到广泛地应用,对疫病防控起到了重 要的作用。由于当前养猪业中猪群中多种病原混合感染病例的增多, 单一病原 PCR 检测方法也表现出自身的不足,需要对一种病料进行多 次 PCR 检测,使得检测时间延长,耗费人力,成本也高。多重 PCR 不仅 保持了普通 PCR 敏感性高和特异性强的优点,而且还具有一个 PCR 反 应就同时检测多种病原,具有简便、快捷、灵敏等优点,能满足生产 上同时对多种疾病进行快速诊断的要求。本研究基于前期对陕西省 部分养殖企业猪群中病毒性病原的调查检测,发现常有 PPV、PRV 和 PCV-2 的单纯或混合感染,为此我们拟建立 PPV、PRV 和 PCV-2 检测 的多重 PCR 方法,以期为生产中这 3 种病原的快速检测提供可用方法。 本研究获得了以下结果:(1)根据 GenBank 中已经发表的 PPV、PRV 和 PCV-2 的全基因序列,利用 DNAStar 和 Oligo6.0 软件,针对病毒的 保守性基因序列,分别设计了检测引物,通过对引物浓度、退火温度、 PCR 组分等条件的优化建立起鉴别 PPV、PRV 和 PCV-2 多重 PCR 方法,方 法敏感性试验表明,PRV、PCV-2 在多重 PCR 反应中的敏感性到达 pg 级,而 PRV 在多重 PCR 反应中的敏感性少了一个数量级,其最低检测 量也达到了 10pg 级。以 PCV-1、牛病毒性腹泻病毒、猪瘟病毒、猪 繁殖障碍与呼吸综合征病毒、猪传染性胃肠炎病毒、大肠埃希菌、 PPV、PRV、PCV-2 为模板分别进行多重 PCR 检测,结果表明只有 PPV、PRV 和 PCV-2 多重 PCR 及单个病毒的 PCR 均扩增出各自的特异 性条带。(2)用建立的方法对采自陕西省部分猪场的 286 份病料及血 样进行病毒检测,从临床健康猪全血样品中 PPV、PRV 和 PCV-2 的检 测结果来看,PCV-2 在正常猪群中有一定比例的存在,同时在个别猪 场也存在 PRV 和 PCV-2 双重感染的现象;从临床患病猪病料中进行检 测发现,PRV 和 PCV-2 混合感染较严重,并有 PPV+PRV+PCV-2 三重感 染发生;多重 PCR 检测结果与单项 PCR 检测结果的符合率达 99%以上,说 明建立的多重 PCR 检测方法可用于临床样品的检测,为这 3 种疫病的 诊断提供依据。 【英文摘要】Porcine parvovirus , porcine pseudorabies and porcine circovirus are three main threat to the development of swine-breeding industry.Beacause sigle or mixed infection of the three virus both can cause diseases which can give rise to reproductive failure or(and) immunosuppression, it provides a convenient chance for secondary infection by other pathogens and enven causes epidemic diseases in pigs wich can lead to tremendous economic losses ,and may arise public health problems. PCR with rapid,specific and sensitive advantages exhibits obvious preponderance in specificit detection of infectious agents in animals since it was invent.And it is applied widely in animal disease diagnosis and pathogen detection which has made important contributions to disease prevention and control. At present,the cases of mixed infection of pathogen are increasing,and common 分子 molecular detection of pathogen has show its own shortage of time consuming ,high cost and labour power when we need detect one specimen repeatedly.Mutiplex PCR not only preserve advantages of routine PCR,but also owns advantages of convenience shortcut and sensitiveness which meet the need of fast diagonosis for multiple diseases in production.We found that sigle or mixed infection of PPV、PRV and PCV-2 is common in the course for the survey of viral pathogen in some breeding enterprises in this research.So we plan to establish the mutiplex PCR for detection of PPV、PRV and PCV-2,in purpose for providing fast detection of the three pathogens an available method .(1)Specific primers for DNA viruses, namely, porcine parvovirus (PPV), pseudorabies virus (PRV), porcine circovirus type 2 (PCV-2),were synthesized according to the published virus conserved gene sequence of PPV, PRV and PCV-2 in GenBank and used by DNAStar and Oligo 6.0 software . We establish a mutiplex PCR method for the detection of PPV, PRV and PCV-2 by optimizing concentration of primers,annealing temperature and component of PCR.Sensibility test shows that the susceptibility of PRV and PCV-2 reach the level of pg.With PCV-1, bovine viral diarrhea virus, classical swine fever virus, porcine reproductive and respiratory syndrome virus, porcine transmissible gastroenteritis virus, Escherichia coli, PPV, PRV, PCV-2 as a template for multiple PCR were detected,the results show that the only PPV, PRV and PCV-2 virus in a single or multiplex PCR were amplified by their specific band.(2)The blood samples collected from 286 pig farms in Shaanxi Province were viral detection by this method. Clinically healthy pigs from a blood sample PPV, PRV and PCV-2 test results indict that PCV-2 in normal pigs have a certain percentage of presence, meanwhile several farms also exist the phenomenon of the PRV and PCV-2 dual infection.Swine from the clinical illness were detected compound, It was serous of PRV+PCV-2 combined infection in diseased pigs,and PPV+PRV+PCV-2 combined infection also were detected in diseased pigs. The coincidence rate of multiplex PCR test and single PCR test is more than 99%, illutstrating that The developed novel multiplex PCR will be a tool used for the detection of clinical samples for diagnosis reference. 【关键词】猪细小病毒 伪狂犬病病毒 猪圆环病毒 2 型 多重 PCR 【英文关键词】Porcine parvovirus(PPV) Pseudorabies virus(PRV) Porcine circovirus type 2 multiplex PCR(mPCR) 【目录】猪细小病毒、伪狂犬病病毒和猪圆环病毒 2 型多重 PCR 检测方法的建立及应用 摘要 6-8 ABSTRACT 8-9 文献综述 12-31 第一章 猪细小病毒、 伪狂犬病病毒和猪圆环病毒 2 型检测方法研究进展 12-26 1.1 猪细小病毒 12-19 1.1.1 概述 12-13 1.1.2 猪细小病毒的特性 13-14 1.1.3 猪细小病毒病 流行情况 14-15 1.1.4 PPV 的检测方法研究 15- 19 1.2 伪狂犬病病毒 19-22 1.2.1 概述 19 1.2.2 伪狂犬病病毒的特性 19-20 1.2.3 伪狂犬病流行情况 20 1.2.4 伪狂犬病病毒检 测方法 20-22 1.3 猪圆环病毒 22-26 1.3.1 概述 22-23 1.3.2 猪圆环病毒的特性 23 1.3.3 猪圆环病毒检测方法 23-26 第二章 聚合酶链 反应(PCR)技术在动物病原检测上的应用 26-31 2.1 PCR 技术概述 26-27 2.1.1 PCR 技术 26 2.1.2 PCR 技术的发展 26-27 2.2 主要的 PCR 技术 27-31 2.2.1 常规 PCR 27 2.2.2 套式 PCR(nested PCR) 27 2.2.3 二温式 PCR 27-28 2.2.4 反转录 PCR (RT PCR) 28 2.2.5 反转录一复合 套式聚合酶链反应 28 2.2.6 反向 PCR 28 2.2.7 竞争 PCR (compete PCR) 28-29 2.2.8 标记 PCR (LP-PCR) 29 2.2.9 定量 PCR 29 2.2.10 玻片 PCR (Slide-PCR 29-30 2.2.11 多重 PCR(Multiplex PCR) 30-31 试验研究 31-45 第三章 猪细小病毒、伪狂犬病病毒和猪圆环病毒 2 型 PCR 检测方 法的建立 31-