非洲猪瘟病毒实时荧光重组酶聚合酶扩增技术(RPA)检测方法的建立

doi:10.16431/j.cnki.1671-7236.2017.11.023

《中国畜牧兽医》 ›› 2017, Vol. 44 ›› Issue (11): 3270-3277.doi: 10.16431/j.cnki.1671-7236.2017.11.023

非洲猪瘟病毒实时荧光重组酶聚合酶扩增技术(RPA)检测方法的建立

哈登楚日亚^1,2, 樊晓旭¹, 赵永刚¹, 王淑娟¹, 张志诚¹, 戈胜强¹, 李林¹, 吴晓东¹, 王志亮¹

1. 中国动物卫生与流行病学中心, 外来病研究中心, 青岛 266032;
2. 内蒙古农业大学兽医学院, 呼和浩特 010018

收稿日期:2017-03-14 出版日期:2017-11-20 发布日期:2017-11-21
通讯作者: 王志亮 E-mail:wangzhiliang@cahec.cn
作者简介:哈登楚日亚(1990-),男,内蒙古正蓝旗人,硕士生,研究方向:非洲猪瘟,Tel:0532-87839158;E-mail:huisheng888@sina.com;樊晓旭(1984-),男,辽宁昌图人,博士,助理研究员,研究方向:外来动物疫病;E-mail:fanxiaoxu@cahec.cn
基金资助:
"十三五"国家重点研发计划（2016YFD0501104）

Establishment of a Real-time Fluorescent Recombinase Polymerase Amplification (RPA) for the Detection of African Swine Fever Virus

HADENG Chu-riya^1,2, FAN Xiao-xu¹, ZHAO Yong-gang¹, WANG Shu-juan¹, ZHANG Zhi-cheng¹, GE Sheng-qiang¹, LI Lin¹, WU Xiao-dong¹, WANG Zhi-liang¹

1. National Research Center for Exotic Animal Disease, China Animal Health and Epidemiology Center, Qingdao 266032, China;
2. College of Veterinary Medicine, Inner Mongolia Agricultural University, Hohhot 010018, China

Received:2017-03-14 Online:2017-11-20 Published:2017-11-21

摘要/Abstract

摘要：

非洲猪瘟（African swine fever，ASF）是由非洲猪瘟病毒（African swine fever virus，ASFV）感染引起的一种急性、热性、高度接触性传染病，给养猪业造成了严重的经济损失。本研究利用重组酶聚合酶扩增技术（RPA），针对ASFV B646L （p72）基因设计了3组引物和探针，并进行筛选、反应条件优化、灵敏性、特异性和重复性试验，建立了实时荧光RPA方法。结果显示，该方法在39℃、20 min内即可检测10个拷贝的DNA分子，且与猪瘟病毒、猪圆环病毒2型、猪细小病毒、猪伪狂犬病病毒均无交叉反应，检测5.5×10⁶至5.5×10⁰拷贝/μL共7个稀释度样品在各时间点的荧光强度，变异系数范围在0.38%~28.30%。该等温、快速扩增方法可用于ASFV的定性检测，为中国ASFV感染的早期诊断提供技术支持，对疫情暴发后相应控制方案的制定具有重要意义。

关键词: 非洲猪瘟病毒(ASFV); 实时荧光重组酶聚合酶扩增(RPA); 灵敏性; 特异性; 重复性

Abstract:

African swine fever (ASF), which caused by African swine fever virus (ASFV), is an acute, highly contagious disease characterized by high fever. It leads to serious economic losses in pig industry. 3 assemblies of primers and probes targeting were designed to amplify ASFV B646L (p72) gene using recombinase polymerase amplification (RPA) technology. The Real-time fluorescent RPA method was established after screening of primers and probes, optimization of reaction conditions, tests of sensitivity, specificity and repeatability. The results showed that the method could detect 10 copies of DNA within 20 min at 39℃. No cross-reaction was found when testing swine fever virus, porcine circovirus type 2, porcine parvovirus, pseudorabies virus. According to the fluorescence intensity from 5.5×10⁶ to 5.5×10⁰ copies/μL at each time point, the coefficient of variation was 0.38% to 28.30%. In conclusion, this method could be used for the qualitative detection of ASFV pathogen, which might provide technical support for the early diagnosis of ASFV infection in China, and was also of great significance for the development of corresponding control measures.

Key words: African swine fever virus (ASFV); Real-time fluorescence recombinase polymerase amplification (RPA); sensitivity; specificity; repeatability

中图分类号:

S852.65⁺1

哈登楚日亚, 樊晓旭, 赵永刚, 王淑娟, 张志诚, 戈胜强, 李林, 吴晓东, 王志亮. 非洲猪瘟病毒实时荧光重组酶聚合酶扩增技术(RPA)检测方法的建立[J]. 《中国畜牧兽医》, 2017, 44(11): 3270-3277.

HADENG Chu-riya, FAN Xiao-xu, ZHAO Yong-gang, WANG Shu-juan, ZHANG Zhi-cheng, GE Sheng-qiang, LI Lin, WU Xiao-dong, WANG Zhi-liang. Establishment of a Real-time Fluorescent Recombinase Polymerase Amplification (RPA) for the Detection of African Swine Fever Virus[J]. , 2017, 44(11): 3270-3277.

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非洲猪瘟病毒实时荧光重组酶聚合酶扩增技术(RPA)检测方法的建立

Establishment of a Real-time Fluorescent Recombinase Polymerase Amplification (RPA) for the Detection of African Swine Fever Virus

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