PRRSV荧光原位杂交检测法的建立及临床应用

发布时间：2019-01-22 16:19

【摘要】：猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)以引起仔猪、保育猪呼吸系统障碍以及母猪繁殖障碍为特征,给养猪业带来巨大的经济损失。PRRSV为单链RNA病毒,属于动脉炎病毒科,含有9个开放阅读框。病毒主要分为两个亚型:北美洲型VR-2332和欧洲型LV。2006年中国爆发了一场规模巨大的“猪高热症”,引起该病的病毒不同于之前的PRRSV,其致病力强,以高热、高致病性和高致死率为特征,经过基因分析确定,这类型的病毒为VR-2332的变异毒株,又称HP-PRRSV。HP-PRRSV病毒在Nsp2当中相比于经典株有90个碱基缺失,并且该突变能够稳定遗传。本实验首先利用Hu N4毒株通过滴鼻的方法感染仔猪,建立病毒模型。在第5、7、14天分别剖杀实验组与对照组样本,固定组织,制成石蜡切片。通过HE染色与免疫荧光组织化学法观察组织学变化,检测病毒分部。分析Nsp2上稳定的序列合成寡聚核苷酸荧光标记探针,通过荧光原位杂交法检测相应组织样品,并且对黑龙江地区50个病例进行检查。实验结果显示,机体肺脏以及淋巴结最先出现病变。肺部出血、水肿、充血严重,出现实变和坏死灶,淋巴结水肿至正常的2-10倍,出现针尖样出血。肝脏和脾脏出现不同程度的水肿和出血。肾脏肿胀或者萎缩,有出血点。组织学观察可看到典型的间质性肺炎,淋巴结和脾脏淋巴细胞大量减少并且坏死,可观察到大量巨噬细胞,有单核细胞和炎性细胞的浸润。免疫荧光组织化学可观察病毒在机体各个器官的分布规律,结果表明感染第五天,可在淋巴结和肺脏、脾脏检测到阳性信号,第七天可从肾脏、肝脏看到明显病毒颗粒,但是信号较少。心脏、胰腺在整个实验过程中均呈现出阴性反应。应用荧光原位杂交法检测各个组织,结果显示阳性信号主要分布于肺脏的肺泡间隔当中的巨噬细胞以及血管内皮细胞,淋巴结当中的淋巴小结以及脾脏当中的淋巴细胞与巨噬细胞。肝脏阳性信号主要出现于窦状隙,肾脏主要分布于肾小球以及肾间质。本实验建立荧光原位杂交法与免疫荧光法和病理组织学观察结果进行比较,发现原位杂交与免疫荧光结果相符,两者均与病理组织学情况一致。
[Abstract]:Porcine Reproductive and Respiratory Syndrome virus (Porcine reproductive and respiratory syndrome virus,PRRSV), characterized by respiratory disorders in piglets, conservation of respiratory disorders in pigs and reproductive disorders in sows, has brought huge economic losses to the pig industry. PRRSV is a single-stranded RNA virus. Belongs to the arteritis virus family, contains 9 open reading boxes. The virus is divided into two subtypes: North American type VR-2332 and European type LV.2006. A huge outbreak of "swine hyperthermia" occurred in China in 2003. The virus causing the disease is different from the previous PRRSV, which is highly virulent and highly febrile. The characteristics of high pathogenicity and high mortality are identified by gene analysis. This type of virus is a variant strain of VR-2332, which is also called HP-PRRSV.HP-PRRSV virus with 90 base deletions compared with classical strains in Nsp2. And the mutation can be inherited stably. In this experiment, Hu N4 strain was first used to infect piglets by nasal drip, and the virus model was established. The samples of the experimental group and the control group were dissected and fixed on the 5th day, and the paraffin sections were made. The histological changes were observed by HE staining and immunofluorescence histochemistry. The stable sequence-synthesized oligonucleotide fluorescent labeled probes on Nsp2 were analyzed. The corresponding tissue samples were detected by fluorescence in situ hybridization and 50 cases in Heilongjiang province were examined. The results showed that lung and lymph nodes were the first lesions. Pulmonary hemorrhage, edema, severe hyperemia, actual changes and necrosis, lymph node edema to normal 2-10 times, needle-like bleeding. Various degrees of edema and bleeding occurred in the liver and spleen. Kidney swelling or atrophy, with bleeding spots. Histologically it was observed that typical interstitial pneumonia lymph node and spleen lymphocytes were decreased and necrotic a large number of macrophages and infiltration of monocytes and inflammatory cells could be observed. Immunofluorescence histochemistry could observe the distribution of virus in various organs of the body. The results showed that on the fifth day of infection, positive signals could be detected in lymph nodes, lungs, spleen, and obvious viral particles could be seen in the kidney and liver on the seventh day. But the signal is less. The heart and pancreas showed negative reactions throughout the experiment. Fluorescence in situ hybridization was used to detect various tissues. The results showed that the positive signals were mainly distributed in macrophages and vascular endothelial cells in the alveolar septum of the lung. Lymphatic nodules in lymph nodes and lymphocytes and macrophages in spleen. Liver positive signal mainly appeared in sinusoid space, kidney mainly distributed in glomeruli and renal interstitial. The results of fluorescence in situ hybridization were compared with those of immunofluorescence and histopathological observation. The results of in situ hybridization were consistent with those of immunofluorescence, and both of them were consistent with histopathology.
【学位授予单位】：东北农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.65

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