摘要：在南京市两家博研宠物医院出现症状为发热、血便、虚弱或临床检测为疑似为细小病毒（Canine Parvovirus type 2, CPV-2）感染的病犬中，采肛拭子40份。用猫肾传代细胞（F81）接毒观察病变，40个样品中有19个样品出现病变，将病变细胞提取DNA，进行PCR和凝胶电泳之后将出现阳性条带的6个样品送至生工生物工程（上海）股份有限公司，测序并比对后结果证明该6个样品均为CPV-2a亚型，将分离到并测定的序列用MEGA 5软件进行遗传进化分析，结果显示在南京博研宠物医院附近地区的犬细小病毒基因型并没有发生突变，该地区的犬细小病毒仍在可控范围之内。50240

毕业论文关键词：犬细小病毒；分离；鉴定；进化分析

The separation，identification and genetic analysis of the Canine Parvovirus type 2 of two pet hospitals in Nanjing area

Abstract: Forty cloacal swabs from some sick dogs with clinical manifestations of elevated body temperature, blood stools, weakness and clinical examinations that were suspected of Canine Parvovirus type 2（CPV-2) were collected from two BoYan pet hospitals. Then the viruses cultured in Feline kidney 81 cell (F81) to observe the cytopathic effect (CPE), 19 samples appeared lesions in 40 samples. Then extracting the DNA from cytopathic cells, with the experiment of PCR and gel electrophoresis and then six positive strip samples were sent to the Sangon Biotech (Shanghai) sequencing company. The sequencing results revealed that there were 6 sequences belong to CPV-2a, then using the sequencing results and software MEGA5 to analyze the genetic evolution. Results shown that there were no mutation of Canine Parvovirus genes nearby BoYan pet hospitals, virus infection is still under control.

Keywords: Canine Parvovirus type 2; separation; identification; evolutionary analysis

目  录

摘要 1

关键词 1

Abstract 1

Keywords 1

引言 1

1.国内外研究概况 1

2.本研究的目的 2

宠物医院采样学习 2

1.材料 2

2.实验方法步骤 3

3.实验结果 3

犬细小病毒的分离鉴定及遗传进化分析 4

1.实验材料 4

1.1.主要实验仪器 4

1.2.其他主要材料 4

1.3.细胞 4

1.4.主要试剂 4

2.实验步骤 4

2.1.样品的过滤分离 4

2.2.细胞传代和接毒 5

2.2.1.细胞的培养以及传代 5

2.2.1.1.复苏 5

2.2.1.2.传代 5

2.3.提取DNA 5

2.4.DNA浓度检测 6

2.5.引物以及PCR反应条件的设计 6

2.6.PCR检测 6

2.7.凝胶电泳 7

2.8.高保真酶PCR 7

2.9.凝胶电泳 7

2.10.送测