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| 含非洲猪瘟病毒核酸序列病毒样颗粒的研制及其在检测方法中的应用 | |
| 引用本文： | 冯春燕,杜方原,刘丹丹,Pershin Andrey,王彩霞,张永宁,吴绍强,林祥梅.含非洲猪瘟病毒核酸序列病毒样颗粒的研制及其在检测方法中的应用[J].中国畜牧兽医,2017,44(11):3287-3293. |
| 作者姓名： | 冯春燕 杜方原 刘丹丹 Pershin Andrey 王彩霞 张永宁 吴绍强 林祥梅 |
| 作者单位： | 1. 中国检验检疫科学研究院动物检疫研究所, 北京 100176; 2. 俄罗斯联邦兽医研究所, 尤里耶韦茨 600900 |
| 基金项目： | 十三五科技支撑计划（2013BAD12B00）；国家质检总局科研基金资助项目（2015IK310） |
| 摘 要： | 非洲猪瘟（African swine fever，ASF）是由非洲猪瘟病毒（African swine fever virus，ASFV）引起的烈性传染病，为了保证其检测结果的准确性和可靠性，需要研制试剂盒中使用的阳性标准质控品。本试验旨在研制含ASFV核酸序列的病毒样颗粒，并探究其在检测方法中的应用。首先扩增p72基因的全长片段，利用昆虫杆状病毒系统，包装出含有p72基因的ASF DNA病毒样颗粒。为了进一步验证该病毒样颗粒在应用中的可靠性，本研究将病毒样颗粒与ASF的组织毒及细胞毒同时进行DNA核酸提取，进行实时荧光定量PCR。结果表明，本研究制备的病毒样粒子能很好的取代ASFV在实时荧光定量PCR检测方法中作为阳性质控品，且能对核酸提取过程进行质控，实时荧光定量PCR检测试剂盒中，病毒样粒子的最低包装浓度为102 TCID50。进一步研究发现该病毒样颗粒也适用于普通PCR及LAMP检测方法中，最低浓度分别为103和101 TCID50。本试验结果将为规范ASF检测方法，促进ASF检测方法的转化应用及保证检测结果的准确度和可靠性提供科学依据。 |
| 关 键 词： | 非洲猪瘟 参考物质 病毒样颗粒 检测方法 |
| 收稿时间： | 2017-05-24 |
Development of Virus-like Particles Containing African Swine Fever Virus Nucleic Acid Sequence and Its Application in Detection Method |
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| FENG Chun-yan,DU Fang-yuan,LIU Dan-dan,Pershin Andrey,WANG Cai-xia,ZHANG Yong-ning,WU Shao-qiang,LIN Xiang-mei.Development of Virus-like Particles Containing African Swine Fever Virus Nucleic Acid Sequence and Its Application in Detection Method[J].China Animal Husbandry & Veterinary Medicine,2017,44(11):3287-3293. | |
| Authors: | FENG Chun-yan DU Fang-yuan LIU Dan-dan Pershin Andrey WANG Cai-xia ZHANG Yong-ning WU Shao-qiang LIN Xiang-mei |
| Institution: | 1. Institute of Animal Quarantine, China Institute of Inspection and Quarantine, Beijing 100176, China; 2. All-Russia Research Institute of Animal Health, Yuryevets 600900, Russia |
| Abstract: | African swine fever (ASF) is an infectious disease caused by the African swine fever virus (ASFV). In order to ensure the accuracy and reliability of the test results,it is necessary to develop positive standard control products used in the kit. The study was aimed to develop the virus-like particles containing African swine fever virus (ASFV) nucleic acid sequence and its application in detection method. Fristly,the full-length gene fragment of p72 gene was amplified, and the ASF DNA virus-like particles containing p72 gene was constructed using insect baculovirus system. In order to further validate the reliability of the virus-like particles in the application of the method,DNA nucleic acid was extracted simultaneously with the cultured virus and infected tissue sample and applied in the Real-time quantitative PCR method. The results showed that the virus-like particles prepared by this study could replace the ASFV as a positive control product in the Real-time quantitative PCR method, and could act as quality control during nucleic acid extraction process. In the fluorescent PCR detection kit, the lowest packaging concentration of virus-like particles was 102 TCID50. Further studies had shown that the virus-like particles were also suitable for common PCR and LAMP detection methods, the minimum concentration were 103 and 101 TCID50, respectively. The results of this study would be important for the ASF detection method, promoting the application of the method and ensuring the accuracy and reliability of the test results. |
| Keywords: | African swine fever (ASF) reference substance virus-like particle detection methods |
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