施马伦贝格病毒实时荧光RT-RAA快检方法的建立

doi:10.11843/j.issn.0366-6964.2024.08.045

畜牧兽医学报 ›› 2024, Vol. 55 ›› Issue (8): 3740-3744.doi: 10.11843/j.issn.0366-6964.2024.08.045

• 研究简报 • 上一篇

施马伦贝格病毒实时荧光RT-RAA快检方法的建立

陈嘉仪(), 黄晓琪, 王炜杰, 莫竣喻, 王苏艳, 丹珍翁姆, 陈劲松, 张瑞, 周泷, 李彦敏*(), 张志东*()

西南民族大学畜牧兽医学院，成都 610041

收稿日期:2023-09-04 出版日期:2024-08-23 发布日期:2024-08-28
通讯作者: 李彦敏,张志东 E-mail:549480733@qq.com;liyanmin@swun.edu.cn;zhangzhidong@swun.edu.cn
作者简介:陈嘉仪(2002-)，女，湖南永州人，本科，主要从事动物病毒检测技术研究，E-mail：549480733@qq.com
基金资助:
西南民族大学“双一流”项目(XM2023012);四川省自然科学基金项目(面上项目)(22NSFSC2548);2022年大学生创新创业训练计划项目(X202210656199);西南民族大学引进高层次人才科研启动金资助项目(16011211013)

Development of a Real-time RT-RAA Assay for Rapid Detection of Schmallenberg Virus

Jiayi CHEN(), Xiaoqi HUANG, Weijie WANG, Junyu MO, Suyan WANG, Wengmu DANZHEN, Jinsong CHEN, Rui ZHANG, Long ZHOU, Yanmin LI*(), Zhidong ZHANG*()

College of Animal Husbandry and Veterinary Medicine, Southwest Minzu University, Chengdu 610041, China

Received:2023-09-04 Online:2024-08-23 Published:2024-08-28
Contact: Yanmin LI, Zhidong ZHANG E-mail:549480733@qq.com;liyanmin@swun.edu.cn;zhangzhidong@swun.edu.cn

摘要/Abstract

摘要：

旨在建立一种快速检测施马伦贝格病毒(SBV)的方法。本研究对于SBV S基因，设计特异性引物和探针，通过优化反应条件，建立SBV反转录重组酶介导核酸恒温扩增(RT-RAA)检测方法。结果显示，该方法在38 ℃条件下，8 min内即可特异性检出SBV，与口蹄疫病毒(FMDV)、小反刍兽疫病毒(PPRV)、非洲马瘟病毒(AHSV)、塞内卡谷病毒(SVV)、水疱性口炎病毒(VSV)无交叉反应，其敏感性为10³拷贝/反应，且批内和批间重复性变异系数均小于5%。对模拟阳性和阴性绵羊样品的检出率为100%，与SBV的RT-qPCR检测结果一致。本研究成功建立了一种快速、敏感、特异的SBV实时荧光RT-RAA方法，为SBV防控提供新技术手段。

关键词: 施马伦贝格病毒, 重组酶介导核酸恒温扩增方法, 快速检测

Abstract:

The aim of this study was to establish a rapid detection method of Schmallenberg virus (SBV). A real-time reverse transcription recombinase acid amplification (RT-RAA) was developed with specific primers and probes based on of SBV S gene. The results showed that, through optimizing experimental conditions, the assay could detect SBV specifically within 8 minutes at 38 ℃. There was no cross-react with foot-and-mouth disease virus(FMDV), pestedes petites ruminants virus(PPRV), African horse distemper virus(AHSV), Seneca valley virus(SVV), or vesicular stomatitis virus(VSV). The detection of limit(LOD)was 10³ copies/reaction. At the same time, the co-efficient of variations in intra- and inter-assay were both less than 5%. The detection rate for the spinked positive samples and negative sheep samples was 100%, which was consistent with SBV RT-qPCR results. This study successfully established a fast, sensitive, and specific real-time RT-RAA assay for detection of SBV, which provided a new technical method for prevention and control of SBV infection.

Key words: Schmallenberg virus, recombinase-acid amplification, rapid detection

中图分类号:

S852.65

陈嘉仪, 黄晓琪, 王炜杰, 莫竣喻, 王苏艳, 丹珍翁姆, 陈劲松, 张瑞, 周泷, 李彦敏, 张志东. 施马伦贝格病毒实时荧光RT-RAA快检方法的建立[J]. 畜牧兽医学报, 2024, 55(8): 3740-3744.

Jiayi CHEN, Xiaoqi HUANG, Weijie WANG, Junyu MO, Suyan WANG, Wengmu DANZHEN, Jinsong CHEN, Rui ZHANG, Long ZHOU, Yanmin LI, Zhidong ZHANG. Development of a Real-time RT-RAA Assay for Rapid Detection of Schmallenberg Virus[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(8): 3740-3744.

图/表 2

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引物/探针 Primer/probe	引物序列(5′→3′) Primer sequences (5′→3′)
SBV-RAA-P	TGCTGAGGTTAAGGGATGCACCTGGGCCGA (FAM-dT)G(THF)T(BHQ1-dT) ATACAATGTATCTTG-P
SBV-RAA-F1	TACATGCAAGGCTAGTGTCCTCAAACTAGCTGAAG
SBV-RAA-F2	GCAAGGCTAGTGTCCTCAAACTAGCTGAAGCTAGT
SBV-RAA-F3	TAGTGTCCTCAAACTAGCTGAAGCTAGTGCTCAGA
SBV-RAA-R1	CAATAACTAGTGGATAGAAGTCAAAAGCATCAAGG
SBV-RAA-R2	CAAAAGCATCAAGGAACATTTCGGCCCCAGGTG
SBV-RAA-R3	AGTGGATAGAAGTCAAAAGCATCAAGGAACATTTC

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施马伦贝格病毒实时荧光RT-RAA快检方法的建立

Development of a Real-time RT-RAA Assay for Rapid Detection of Schmallenberg Virus

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