猪圆环病毒2型的分离鉴定及其对BALBc小鼠的致病性研究(5)

江苏部分地区猪圆环病毒2型的分离鉴定及其对BALB/c小鼠的致病性研究

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联免疫吸附实验[94]（cELISA），具有更高的敏感性和特异性。

6.5 聚合酶链式反应（Polymerase Chain Reaction，PCR）

聚合酶链式反应是一种使特定的DNA片段放大扩增的分子生物学技术，它可看作是体外进行的的特殊DNA复制，PCR的能将微量的DNA大幅扩增。PCR(聚合酶链式反应）的原理是利用DNA在95°C高温时变性变成单链，低温（经常是60°C左右）时引物与单链按碱基互补配对的原则结合，再将温度调至72°C左右DNA聚合酶沿着DNA链(5'-3')的方向合成互补链。PCR的关键是引物的设计，引物要有一定的特异性，而退火的温度也很关键。PCR可以用来区分PCV1和PCV2，并且用来诊断PCV相关的疾病。PCR对检测最低含量的病毒核酸有独特的敏感性。可以用来鉴定皮肤、肾、淋巴结、脾、扁桃体、肝和肺组织等样品是否存在PCV2[65]。大多数的PMWS 是由PCV2 和PPRSV的混合感染引起的，通过PCR、ISH、IHC和IIFA可以区分出PCV2和PRRSV病毒[95-98]。PCR的方法检测核酸比原位杂交（ISH）更为敏感[65]。

7 PCV2相关的疾病防治（PCVAD）防治

PCV2相关的疾病防治非常困难，甚至在严格隔离的猪场也有PCVAD的流行暴发，猪细小病毒(PPV)的疫苗并不能降低感染猪患病的严重性[99]，但是猪细小病毒与猪丹毒的两联苗可以保护PCV2感染的猪不会发生生殖障碍[100]。

接种PCV2的疫苗可以很大程度的减少PCV2感染带来的损失[101]。良好的管理条件也是严格预防疾病的关键。减轻压力、注意环境卫生、各年龄段猪分开饲养、严格的―全进全出‖的饲养，定期消毒能够有效的控制PCV2的感染。但即使是严格控制用量的血清学免疫疗法也可能是无效的，因为病毒对环境有很强的适应力和抵抗力。

预防和治疗猪的细菌性的感染以及能够引起混合感染的相关的病毒也是一种有效防止PCV2疾病的方法。猪细小病毒（PPV）或猪蓝耳病毒（PPRSV）与PCV2的混合感染可以加重PCVAD的病情，减少这些危险因子的感染对控制PCVAD十分重要。增加猪群间的空隙、定期进行寄生虫的治疗接种[102]以及猪萎缩性鼻炎疫苗等[103,104]措施能有效减少这些危险因子感染，同时做好其他疫病的免疫接种，使猪群的免疫水平提高[105]。

快速诊断、隔离和淘汰病猪[106]，同时提供良好的管理和饲养的条件，是目前唯一可以减少PCV2的感染带来的损失的方法。

8 研究目的和意义

本研究通过采集扬州大学动物医院的病死猪和扬州市规模化屠宰场的组织样品，通过PCR检测样品中PCV1、PCV2、PPV、PRV、PRRSV和CSFV六种常见病毒的流行情

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扬州大学硕士论文

况，统计本地区六种病毒的感染情况，并用Dulac细胞对PCV2检测阳性的组织样品进行病毒分离，对分离到的PCV2病毒测序并绘制遗传进化树。为该地区的流行病学调查和猪肉类食品安全提供依据。

近年来，全国PCV2感染引起的疾病在全国呈逐年上升趋势，给养猪业造成直接或间接的巨大经济损失。对PCV2的致病机制研究有十分重要的意义。关于PCV2致病性的研究虽多，但在小鼠动物模型上研究尚少。本研究在成功用PCV2感染猪模型的基础上尝试研究PCV2对BALB/c小鼠的致病性，为PCV2感染小鼠模型提供依据，对PCV2的分子致病机制奠定研究基础。

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