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摘要：猪萨佩罗病毒可以引起猪繁殖系统、神经系统以及皮肤系统等多重复合型疾病，主要经粪-口途径传播，如果在临床上跟其他种类如猪瘟病毒、猪细小病毒、猪繁殖与呼吸综合征病毒之类猪致病病原混合感染将引起更为严重的病症[1]。实验依照已经在NCBI发表的中国地区猪萨佩罗病毒基因序列设计合成了一对特异性上下游引物，利用PCR扩增的方法调查该病毒在大陆猪场的感染和流行情况，并摸索了RT-PCR检测的最优方法。根据实验结果，本次试验中所优化的RT-PCR 检测方法仅对猪萨佩罗病毒具有特异性扩增并在紫外线下显示清晰的条带，对其它类型的猪的临床常见病毒如猪瘟等均呈阴性，因此可以认为该PCR条件具有较高的特异性；而猪萨佩罗病毒的最低检测限为4×103 拷贝/μL，因此可以认为该PCR条件有较高的敏感性；利用摸索的PCR条件对2014 到2016 年年间采自我国的数十家猪场的345份临床样品进行检测，临床猪萨佩罗病毒感染的感染率为8.12 % (28/345)，表明我国猪场中存在着比较普遍的猪萨佩罗病毒感染。此外，对实验室分离的猪萨佩罗病毒全基因依据长度分为751个片段分别PCR扩增，通过测序拼接成全序列，再利用序列分析软件对其基因进行序列分析。实验结果表明，实验室分离得到的毒株（PSV-JS2016）与已知中国株(湖南株)分属于同一个分支，说明目前我国的猪萨佩罗毒株的基因序列相对统一保守。而包括江苏株和湖南株在内的中国分离株与韩国株、英国株、美国株等处在明显不同的分支，又说明我国猪萨佩罗病毒的毒株可能存在着相对独立的进化。27475
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毕业论文关键词：猪萨佩罗病毒；RT-PCR；检测；基因测序分析
Development and application of a RT-PCR assay for detection of porcine Sapelovirus
Abstract：Porcine sapelovirus is mainly spread by the cast-mouth way route, which is one of the most common porcine viruses that cause multiple problems on respiratory system, nervous system, reproductive system and skin barrier systems. If it mixed infect with other kinds of porcine viruses(PPV,PRRSV,CSFV and so on) in clinical will cause serious illness. We designed a pair of specific primer according to the published gene sequence of porcine Sapelovirus (PSV) and build an identification method by RT-PCR in order to evaluate the infection and epidemic of the virus in our country. According to the results of test, the PCR shows that it only has specific amplification on PSV and negative phenomenon on other mainly porcine viruses like PPV so we can say that the specificity is pretty obvious; the lowest detectable limit is 4×103 copy/μL which sensibility is highly as expected; we tested 345 samples between 2014 and 2016 from dozens of pig farms shows that the positive rate is 8.12%(28/345) which means there is general infection among pigs in our country. Beside, we test the completed nucleotide sequence of PSV by PCR and analyze the sequence use different softwares such as DNAStar and MEGA. It shows that the strain we got belong to the big branch of PSV in China, so we can get the conclusion that the sequence of Chinese PSV is conserved. But all the stains from China is very different with stains from Korea and America it also means Chinese PSV may evolved independent.
Key words: porcine Sapelovirus; RT-PCR; detection；gene sequencing analysis
目录
摘要1
关键词1
Abstract1
Key words 1
前言2
1 材料与方法 2
1.1 主要实验材料 2
1.1.1 病料样品 2
1.1.2 培养基及抗生素配置 3
1.1.3 主要试剂 3
1.2 引物的设计与合成3
1.3 病料样品的处理及模板的制备3
1.4 RT- PCR 检测方法的优化3
1.5 特异性实验 3
1.6 敏感性实验 3
1.7 RT- PCR 方法的可靠性检测 4