兔出血症病毒的Taq-Man实时荧光定量RT-PCR方法建立及家兔和乳鼠感染检测

发布时间：2018-04-09 05:23

本文选题：兔瘟病毒(RHDV)　切入点：Taq-Man探针　出处：《四川农业大学》2016年硕士论文

【摘要】：兔病毒性出血症(rabbit hemorrhagic disease, RHD)又称兔瘟,是由兔出血症病毒(rabbit hemorrhagic disease virus, RHDV)引起的一种感染成年兔的急性、烈性、高发病率和高致死率的传染病,严重危害着世界养兔业的发展。由于RHDV尚无稳定的培养细胞,这影响着RHDV的细胞侵染机理和防控等方面的研究,加之RHDV近年新的变异毒株(RHDV2)的出现和流行,这为RHD的防治带来了新的困难。因此本文将建立的Taq-Man实时荧光定量RT-PCR检测方法对新出现的RHDV的变异株也进行了特异性验证。同时进行了RHDV的家兔和乳鼠感染检测试验,具体如下：根据兔瘟病毒VP60蛋白基因的序列设计了一对引物,克隆出986bp的RHDV VP60基因片段作为实时荧光定量RT-PCR检测的阳性模板。在克隆阳性模板的目的片段区间,利用Beacon Designer 8软件设计1对引物及1条探针,目的片段为173bp,在探针的5’端标记FAM,3’端标记TAMRA,建立Taq-Man实时荧光定量RT-PCR的检测方法,并对反应条件进行了优化,绘制了标准曲线。通过灵敏度试验和特异性试验表明,建立的Taq-Man实时荧光定量RT-PCR具有较高的灵敏度和良好的特异性,适用于RHDV临床样品的快速准确检测。对市场上买来未免疫过RHDV的10只成年家兔进行RHDV感染,10只家兔3天内死亡,对家兔的主要内脏器官制作并观察了病理切片,同时采集的家兔的主要内脏器官各0.2g,血液200μL进行病毒含量检测,结果显示病死家兔具有典型的兔瘟病理组织学变化,且各内脏器官中均含有RHDV,阳性率为100%,各个器官中含毒量大小依次为血浆,血细胞,肝脏,脾脏,心脏,肾脏,肺脏,肌肉。同时分别对乳鼠进行了背部注射RHDV的体内感染检测和乳鼠主要脏器原代细胞培养后接毒的检测试验,结果显示乳鼠在24h内死亡,死亡乳鼠的各个脏器的RHDV含量检测均为阳性,其中在肾脏和肺脏中的病毒含量较高。培养了乳鼠主要脏器的原代细胞,接种RHDV后,RHDV的检测结果显示,除了乳鼠肾脏原代细胞内有微量RHDV的存在,其他脏器的原代细胞中RHDV的含量可疑。以上的研究为RHDV细胞侵染的机制研究奠定了基础和提供了科学参考。
[Abstract]:Rabbit hemorrhagic disease (RHD), which is caused by rabbit hemorrhagic disease virus (RHDV), is an acute, acute, high morbidity and high mortality infectious disease of adult rabbits, which seriously endangers the development of rabbit breeding in the world.As there are no stable cultured cells in RHDV, this affects the study of cell infection mechanism and prevention and control of RHDV, in addition to the emergence and prevalence of new variant strain RHDV2 of RHDV in recent years, this brings new difficulties to the prevention and treatment of RHD.Therefore, the Taq-Man real-time quantitative RT-PCR detection method was used to detect the new variant strains of RHDV.A pair of primers were designed according to the sequence of VP60 protein gene of rabbit plague virus, and the RHDV VP60 gene fragment of 986bp was cloned as a positive template for real-time quantitative RT-PCR detection.A pair of primers and a probe were designed by using Beacon Designer 8 software in the region of the target fragment of the clone positive template. The target fragment was 173bp. Tamra was labeled at the 5 'end of the probe, and a real-time quantitative RT-PCR detection method for Taq-Man was established.The reaction conditions were optimized and the standard curve was drawn.The sensitivity test and specificity test show that the Taq-Man real-time quantitative RT-PCR has high sensitivity and good specificity and is suitable for rapid and accurate detection of RHDV clinical samples.Ten adult rabbits who had not been immunized with RHDV were infected with RHDV in the market. 10 rabbits died within 3 days. The main visceral organs of the rabbits were made and the pathological sections were observed.At the same time, the main visceral organs and 200 渭 L of blood collected from rabbits were detected for virus content. The results showed that the histopathological changes of rabbit plague were typical in the dead rabbits.RHDV was found in all visceral organs with a positive rate of 100. The virulence in each organ was plasma, blood cells, liver, spleen, heart, kidney, lung and muscle.At the same time, the infection of RHDV injected into the back and the infection of the primary cells of the main organs of the newborn rats were detected respectively. The results showed that the newborn mice died within 24 hours, and the RHDV content of each organ of the dead mice was positive.The virus content in kidney and lung is higher.Primary cells of main organs of newborn rats were cultured. After inoculation with RHDV, the results showed that there was a small amount of RHDV in the primary cells of the kidney, but the content of RHDV in the primary cells of other organs was suspicious.The above studies have laid a foundation for the study of the mechanism of RHDV cell infection and provided scientific reference.
【学位授予单位】：四川农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S858.291

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