非洲马瘟病毒和西尼罗病毒双重TaqMan荧光定量RT-PCR检测方法的建立及应用

doi:10.11843/j.issn.0366-6964.2024.12.048

畜牧兽医学报 ›› 2024, Vol. 55 ›› Issue (12): 5873-5879.doi: 10.11843/j.issn.0366-6964.2024.12.048

非洲马瘟病毒和西尼罗病毒双重TaqMan荧光定量RT-PCR检测方法的建立及应用

钱佳豪¹^,²(), 刘丹¹, 周师众¹, 张博源¹, 高建帅¹, 蒋卉¹, 范学政¹, 张广智¹, 丁家波¹, 王春凤²^,*(), 沈青春¹^,*()

1. 中国农业科学院北京畜牧兽医研究所农业农村部动物生物安全风险预警及防控重点实验室(北方), 北京 100193
2. 吉林农业大学动物医学院, 长春 130118

收稿日期:2023-12-12 出版日期:2024-12-23 发布日期:2024-12-27
通讯作者: 王春凤,沈青春 E-mail:1318672013@qq.com;wangchunfeng@jlau.edu.cn;shenqingchun@caas.cn
作者简介:钱佳豪(1998-), 浙江金华人, 主要从事人畜共患病研究, E-mail: 1318672013@qq.com
基金资助:
外来动物疫病传入溯源精准诊断技术与产品研究(2022YFD1800503)

Establishment and Application of Dual TaqMan Fluorescence RT-PCR Detection Method for African Horse Sickness Virus and West Nile Fever Virus

QIAN Jiahao¹^,²(), LIU Dan¹, ZHOU Shizhong¹, ZHANG Boyuan¹, GAO Jianshuai¹, JIANG Hui¹, FAN Xuezheng¹, ZHANG Guangzhi¹, DING Jiabo¹, WANG Chunfeng²^,*(), SHEN Qingchun¹^,*()

1. Key Laboratory of Animal Biosafety Risk Prevention and Control (North), Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, China
2. College of Veterinary Medicine, Jilin Agricultural University, Changchun 130118, China

Received:2023-12-12 Online:2024-12-23 Published:2024-12-27
Contact: WANG Chunfeng, SHEN Qingchun E-mail:1318672013@qq.com;wangchunfeng@jlau.edu.cn;shenqingchun@caas.cn

摘要/Abstract

摘要：

本研究旨在建立一种可同时检测非洲马瘟病毒(African horse sickness virus, AHSV)和西尼罗病毒(West Nile virus, WNV)的双重荧光定量RT-PCR检测方法。根据AHSV VP7基因序列和WNV Poly Protein基因的高度保守区域设计特异性引物和探针，优化反应条件及体系，绘制标准曲线，对其特异性、敏感性和重复性进行测定，并采用该方法对临床样品进行检测验证。结果显示：该方法能够同时检测AHSV和WNV，且特异性良好，与马动脉炎病毒、马疱疹病毒1型、马传染性贫血病毒、马腺疫链球菌、马流感病毒等马病核酸均无交叉反应；敏感性高，AHSV和WNV最低检测限均为10 copies·μL^-1；组内变异系数为0.04%~0.93%，组间变异系数为0.17%~4.83%，重复性良好；使用该方法对30份马全血样品进行检测，结果均为阴性。本试验建立的检测方法对马属动物检疫、非洲马瘟和西尼罗热的监测与防控具有重要意义。

关键词: 双重荧光定量RT-PCR, 非洲马瘟病毒, 西尼罗病毒

Abstract:

This study aimed to develop a dual-fluorescence quantitative RT-PCR detection method capable of simultaneously identifying African horse sickness virus (AHSV) and West Nile virus (WNV). Specific primers and probes were designed based on highly conserved regions of the AHSV VP7 gene and WNV Poly Protein gene. The reaction conditions and system were optimized, standard curves were established, and the method's specificity, sensitivity, and reproducibility were assessed. Clinical sample testing was conducted to validate the method. Results demonstrated that the method could effectively detect both AHSV and WNV with excellent specificity. No cross-reactivity was observed with equine arteritis virus (EAV), equid Herpesvirus-1 (EHV-1), equine infectious anemia virus (EIAV), Streptococcus equi subsp. zooepidemicus (SEZ), equine influenza virus (EIV) and other equine nucleic acids. The method exhibited high sensitivity, with the lowest detection limits for AHSV and WNV both at 10 copies·μL^-1. Intra-assay coefficients of variation ranged from 0.04% to 0.93%, inter-assay coefficients of variation ranged from 0.17% to 4.83%, demonstrating good reproducibility. Testing 30 equine whole blood samples using this method yielded negative results. The detection method established in this study holds significant importance for equine animal quarantine, monitoring, and control of African horse sickness and West Nile fever.

Key words: duplex real-time RT-PCR, African horse sickness virus, West Nile virus

中图分类号:

钱佳豪, 刘丹, 周师众, 张博源, 高建帅, 蒋卉, 范学政, 张广智, 丁家波, 王春凤, 沈青春. 非洲马瘟病毒和西尼罗病毒双重TaqMan荧光定量RT-PCR检测方法的建立及应用[J]. 畜牧兽医学报, 2024, 55(12): 5873-5879.

QIAN Jiahao, LIU Dan, ZHOU Shizhong, ZHANG Boyuan, GAO Jianshuai, JIANG Hui, FAN Xuezheng, ZHANG Guangzhi, DING Jiabo, WANG Chunfeng, SHEN Qingchun. Establishment and Application of Dual TaqMan Fluorescence RT-PCR Detection Method for African Horse Sickness Virus and West Nile Fever Virus[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(12): 5873-5879.

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参考文献 23

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病原 Pathogen	基因 Gene	引物及探针序列(5′→3′) Primer and probe	片段长度/bp Size
AHSV	VP7	F：TTTCAGCAGATGAATATGCAGCCTAT R：ACACTAATGAAAGCGGTGACCGTGCA P：FAM-AGAGCTCTTGTGCTAGCAGC-BHQ1	233
WNV	PolyProtein	F：TACAAYGCYGAYATGATTGA R：ATGCTGATCTTGGCRGTCCACCT P：VIC-TGGGCCTTCTGGTCGTGTTCTTGG-BHQ1	101

病原 Pathogen	稀释倍数/(copies·μL^-1) Dilution	组内 Intra-assay		组间 Inter-assay
病原 Pathogen	稀释倍数/(copies·μL^-1) Dilution	Ct值Ct value (${\bar x}$±s)	变异系数/% CV	Ct值Ct value (${\bar x}$±s)	变异系数/% CV
AHSV	1×10⁹	10.28±0.04	0.39	9.92±0.26	2.57
	1×10⁸	13.45±0.02	0.12	13.34±0.07	0.55
	1×10⁷	16.46±0.01	0.04	16.39±0.05	0.31
	1×10⁶	19.96±0.01	0.07	19.93±0.03	0.17
	1×10⁵	23.34±0.03	0.13	24.29±0.67	2.77
	1×10⁴	26.86±0.09	0.11	27.79±0.66	2.37
	1×10³	30.26±0.03	0.08	31.25±0.70	2.25
	1×10²	33.55±0.12	0.37	34.95±0.99	2.84
	1×10¹	36.74±0.15	0.41	39.43±1.90	4.83
WNV	1×10⁹	8.52±0.07	0.80	8.87±0.25	2.84
	1×10⁸	11.55±0.03	0.23	12.30±0.54	4.36
	1×10⁷	14.43±0.05	0.34	15.30±0.62	4.04
	1×10⁶	18.33±0.02	0.09	19.23±0.63	3.29
	1×10⁵	21.68±0.04	0.19	22.60±0.65	2.88
	1×10⁴	24.88±0.07	0.26	25.80±0.68	2.65
	1×10³	28.40±0.04	0.13	29.42±0.73	2.46
	1×10²	32.18±0.29	0.93	32.99±0.64	1.94
	1×10¹	35.12±0.06	0.16	36.19±0.77	2.11

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非洲马瘟病毒和西尼罗病毒双重TaqMan荧光定量RT-PCR检测方法的建立及应用

Establishment and Application of Dual TaqMan Fluorescence RT-PCR Detection Method for African Horse Sickness Virus and West Nile Fever Virus

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