RT-RAA detection method based on porcine reproductive and respiratory syndrome virus Nsp2 gene

doi:10.6048/j.issn.1001-4330.2025.03.025

Xinjiang Agricultural Sciences ›› 2025, Vol. 62 ›› Issue (3): 748-753.DOI: 10.6048/j.issn.1001-4330.2025.03.025

• Animal Husbandry Veterinarian • Previous Articles Next Articles

RT-RAA detection method based on porcine reproductive and respiratory syndrome virus Nsp2 gene

YU Na¹^,²(), ZHAO Aiyun¹, HUANG Chunyuan², MA Jiamei²^,³^,⁴, ZHANG Ziwei²^,³, FAN Yuexuan²^,³, ZHENG Jiaxin², ZHANG Yan²^,³, LIU Guangliang²^,³^,⁵, QI Meng¹, CAO Zongxi¹^,²^,³()

1. Tarim Animal Disease Diagnosis and Prevention Engineering Laboratory of Xinjiang Production and Construction Corps/College of Animal Science and Technology, Tarim University, Aral Xinjiang 843300, China
2. Hainan Provincial Key Laboratory of Tropical Animal Breeding and Disease Research, Institute of Animal Science and Veterinary Medicine/Hainan Academy of Agricultural Sciences, Haikou 571100, China
3. College of Veterinary Medicine, Xinjiang Agricultural University, Urumqi 830052, China
4. Suzhou Mars Biotechnology Co, Ltd., Suzhou Jiangsu 215021, China
5. Hainan Experimental Animal Research Center, Sanya Research Institute, Hainan Academy of Agricultural Sciences, Sanya Hainan 572000, China

Received:2024-08-13 Online:2025-03-20 Published:2025-05-14
Correspondence author: CAO Zongxi
Supported by:
National Key R&D Program of China(2023YFC3404302);National Key R&D Program of China(2021YFA0805905);Key R&D Project of Hainan Province(ZDYF2023XDNY038);National Natural Science Foundation of China(32060796);Major Science and Technology Program Project of Hainan Province(ZDKJ2021030)

反转录-重组酶介导等温扩增(RT-RAA)快速检测猪繁殖与呼吸综合征

于娜¹^,²(), 赵爱云¹, 黄春媛², 马佳镁²^,³^,⁴, 张紫薇²^,³, 范悦轩²^,³, 郑佳馨², 张艳²^,³, 刘光亮²^,³^,⁵, 齐萌¹, 曹宗喜¹^,²^,³()

1.塔里木大学动物科学与技术学院/新疆生产建设兵团塔里木动物疫病诊断与防控工程实验室,新疆阿拉尔 843300
2.海南省农业科学院畜牧兽医研究所/海南省热带动物繁育与疫病研究重点实验室,海口 571100
3.新疆农业大学动物医学学院,乌鲁木齐 830052
4.苏州天隆生物科技有限公司,江苏苏州 215021
5.海南省农业科学院三亚研究院/海南省实验动物研究中心,海南三亚 572000

通讯作者: 曹宗喜
作者简介:于娜(2000-),女,内蒙古赤峰人,硕士研究生,研究方向为家畜传染病学,(E-mail) 2359101244@qq.com
基金资助:
国家重点研发计划项目(2023YFC3404302);国家重点研发计划项目(2021YFA0805905);海南省重点研发计划项目(ZDYF2023XDNY038);国家自然科学基金项目(32060796);海南省重大科技计划项目(ZDKJ2021030)

Abstract

Abstract:

【Objective】The objective of this study is to establish a rapid detection method for PRRSV Nsp2 gene by reverse transcription recombinase-mediated isothermal amplification (RT-RAA) fluorescence. 【Methods】Primers and probes of classical strains and highly virogenic strains were designed to detect the sensitivity and specificity of RT-RAA.【Results】The results showed that the proposed method had no cross-reactivity with the nucleic acids of swine fever virus (CSFV), pseudorabies virus (PRV Bartha-K61), porcine epidemic diarrhea virus (PEDV Purdue) and porcine infectious gastroenteritis virus (TGEV LJX). The minimum detection limit of this method was 1.71×10¹ copies/μL for highly pathogenic strains and 2.19×10³ copies/μL for classical strains. Compared with the PCR method, the sensitivity and specificity of RT-RAA fluorescence quantification method were 95.5% and 100%, respectively. The sensitivity and specificity of RT-RAA classical strain fluorescence quantification method were 93.0% and 100%, respectively and the two methods were highly consistent. 【Conclusion】The PRRSV RT-RAA fluorescence method established in this study can distinguish two different strains with good specificity and high sensitivity.

Key words: PRRSV; RT-RAA; Nsp2 gene; specificity; sensitivity; highly virulent strains; classical strains

摘要：

【目的】针对PRRSV Nsp2基因建立反转录重组酶介导等温扩增(RT-RAA)荧光法快速检测方法。【方法】设计经典毒株和高致病毒株引物及探针,检测 RT-RAA 的敏感性及特异性等。【结果】方法与猪瘟病毒(CSFV)、伪狂犬病毒(PRV Bartha-K61)、猪流行性腹泻病毒(PEDV Purdue)、猪传染性胃肠炎病毒(TGEV LJX)的核酸均无交叉反应;方法对高致病性毒株最低检出限为1.71×10¹copies/μL,对经典株最低检出限为2.19×10³ copies/μL。高致病毒株方法敏感性为95.5%,特异性为100%;经典株方法敏感性为93.0%,特异性为100%,2种方法具有高度的一致性。【结论】建立的 PRRSV RT-RAA 荧光法可以区分2种不同的毒株并且特异性良好、灵敏度高。

关键词: 猪繁殖于呼吸综合征病毒, RT-RAA, Nsp2 基因, 特异性, 敏感性, 高致病毒株, 经典毒株

CLC Number:

S855

YU Na, ZHAO Aiyun, HUANG Chunyuan, MA Jiamei, ZHANG Ziwei, FAN Yuexuan, ZHENG Jiaxin, ZHANG Yan, LIU Guangliang, QI Meng, CAO Zongxi. RT-RAA detection method based on porcine reproductive and respiratory syndrome virus Nsp2 gene[J]. Xinjiang Agricultural Sciences, 2025, 62(3): 748-753.

于娜, 赵爱云, 黄春媛, 马佳镁, 张紫薇, 范悦轩, 郑佳馨, 张艳, 刘光亮, 齐萌, 曹宗喜. 反转录-重组酶介导等温扩增(RT-RAA)快速检测猪繁殖与呼吸综合征[J]. 新疆农业科学, 2025, 62(3): 748-753.

Figures/Tables 6

Fig.1 Sensitivity of real-time RAA-HTaq1method Notes:1-9: In turn 1.71×109~101 copies/μL 11: Negative control

Fig.2 Sensitivity of real-time RAA-CTaq2 method Notes:1-7: In turn.2.19×107~101 copies/μL8: Negative control

Fig.3 Specificity of real-time RAA-HTaq1 method Notes:1-6: In turn.PRRSV TJ-F92、pMD18T-NSP2-VR2332、CSFV、PRV Bartha-K61、PEDV Purdue、TGEV LJX 7: Negative control

Fig.4 Specificity of real-time RAA-CTaq2 method Notes:1-6: In turn.PRRSV VR2332、pMD18T-NSP2-TJ、CSFV、PRV Bartha-K61、PEDV Purdue、TGEV LJX7: Negative control

Tab.1 Comparisons of RT-RAA fluorescence method and PCR method between RT-RAA fluorescence method and PCR method for the detection of clinical specimens of highly virogenic strains

项目 Items		阳性 Positives NO.	阴性 Negative NO.	总量 Total
RT-RAA 荧光法 RT-RAA fluorescence	阳性	64	0	64
	阴性	3	133	136
		67	133	200

Tab.2 Comparisons table of RT-RAA fluorescence method and PCR method for the detection of clinical specimens by classical strains

项目 Items		阳性 Positives NO.	阴性 Negative NO.	总量 Total
RT-RAA 荧光法 RT-RAA fluorescence	阳性	67	0	67
	阴性	5	128	133
		72	128	200

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RT-RAA detection method based on porcine reproductive and respiratory syndrome virus Nsp2 gene

反转录-重组酶介导等温扩增(RT-RAA)快速检测猪繁殖与呼吸综合征

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