| 猪圆环病毒2型实时荧光定量PCR检测方法的建立 |
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| 引用本文： | 于静,劳秀杰,陈彦永,何小江,代兵,赵阿勇,王晓杜,宋厚辉.猪圆环病毒2型实时荧光定量PCR检测方法的建立[J].浙江农林大学学报,2016,33(2):357-363. |
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| 作者姓名： | 于静 劳秀杰 陈彦永 何小江 代兵 赵阿勇 王晓杜 宋厚辉 |
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| 作者单位： | 1.浙江农林大学 动物科技学院，浙江 临安 311300 |
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| 基金项目： | 浙江省科技厅公益项目（2014C32061）;浙江农林大学人才启动项目（2011FR025，2013FR077）;浙江省自然科学基金资助项目（LQ14C010007）;浙江农林大学大学生科技创新训练计划项目（201301017）;浙江农林大学面上基金项目（2013FK001） |
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| 摘 要： | 猪圆环病毒2型（porcine circovirus 2, PCV2），是感染猪Sus scrofa domestica的一种单链DNA病毒。建立一种快速、灵敏的检测方法，对于PCV2感染猪的筛选和疾病预防非常重要。根据PCV2 ORF2基因保守区，设计了荧光定量聚合酶链式反应（PCR）引物，利用SYBR Green作为荧光染料建立了一种定量检测PCV2的PCR方法。结果表明:该方法具有灵敏度高、特异性强和重复性好的优点，每微升的最低检测限低至101拷贝DNA。利用该方法对34份PCV2阳性临床样本进行检测，检测符合率为100%，明显高于普通PCR方法的50.0%。因此，本研究建立的PCV2实时荧光定量PCR检测方法为该疾病的预防和控制提供一种有效的检测工具。图4表4参26
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| 关 键 词： | 动物学 猪 猪圆环病毒2型 实时荧光定量PCR |
| 收稿时间： | 2015-04-19 |
A real-time PCR method for detection of porcine circovirus 2 |
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| YU Jing,LAO Xiujie,CHEN Yanyong,HE Xiaojiang,DAI Bing,ZHAO Ayong,WANG Xiaodu,SONG Houhui.A real-time PCR method for detection of porcine circovirus 2[J].Journal of Zhejiang A&F University,2016,33(2):357-363. |
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| Authors: | YU Jing LAO Xiujie CHEN Yanyong HE Xiaojiang DAI Bing ZHAO Ayong WANG Xiaodu SONG Houhui |
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| Institution: | 1.College of Animal Science and Technology, Zhejiang A & F University, Lin’an 311300, Zhejiang, China |
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| Abstract: | To develop an accurate and rapid detection method for disease screening and prevention with porcine circovirus type 2 （PCV2）, a single-strand DNA virus that infects pigs, primers targeted to the ORF2 fragment of the PCV2 conserved region were designed. A real time polymerase chain reaction （PCR） method was developed using SYBR Green as a fluorescent dye and serial dilutions of ORF2 recombinant plasmid to construct a standard curve for an absolute quantification. Results showed that the detection limit was obtained with 101 copy DNA per microliter. This method exhibited 1 000 times higher sensitivity than conventional PCR, only specific identification of PCV2, and better repeatability with the less than 2% variation coefficient of intra- or inter-assay experiments. A total of 34 PCV2 positive clinical samples were confirmed using this real time PCR method, demonstrating 100% agreement in comparison with the conventional PCR having only 50% agreement. This real time PCR method could provide a valuable tool for PCV2 prevention and control.［Ch, 4 fig. 4 tab. 26 ref.］ |
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