Establishment and application of a fluorescent assay for reverse transcription recombinase-mediated isothermal amplification of porcine reproductive and respiratory syndrome virus

doi:10.6048/j.issn.1001-4330.2025.05.027

Xinjiang Agricultural Sciences ›› 2025, Vol. 62 ›› Issue (5): 1286-1292.DOI: 10.6048/j.issn.1001-4330.2025.05.027

• Animal Husbandry Veterinarian·Agricultural Eeconomy • Previous Articles Next Articles

Establishment and application of a fluorescent assay for reverse transcription recombinase-mediated isothermal amplification of porcine reproductive and respiratory syndrome virus

ZHANG Ziwei¹^,²(), HUANG Chunyuan², YU Na¹^,³, ZHENG Jiaxin², ZHANG Yan², LIU Guangliang¹^,², CAO Zongxi¹^,²^,³^,⁴()

1. College of Animal Medicine, Xinjiang Agricultural University, Urumqi 830000, China
2. Key Laboratory of Tropical Animal Breeding and Epidemic Disease Research / Haikou Observation Experimental Station of National Data Center of Animal Health, Hainan Province/Institute of Animal Husbandry and Veterinary Medicine, Hainan Academy of Agricultural Sciences, Haikou 571100, China
3. Tarim Animal Epidemic Disease Diagnostic and Prevention and Control Engineering Laboratory of Xinjiang Production and Construction Corps, Faculty of Animal Science and Technology, Tarim University, Aral Xinjiang 843300,China
4. Samya Research Institute, Hainan Academy of Agricultural Sciences (Hainan Research Centre of Laboratory Animal),Sanya Hainan 572000,China

Received:2024-10-16 Online:2025-05-20 Published:2025-07-09
Correspondence author: CAO Zongxi
Supported by:
National Natural Science Foundation of China(32060796);Hainan Provincial Key R & D Project(ZDYF2023XDNY038);National Key R & D Program Project(2023YFC3404302);National Key R & D Program Project(2021YFA0805905);Major Science and Technology Program Project in Hainan Province(ZDKJ2021030)

猪繁殖与呼吸综合征病毒的逆转录重组酶介导等温扩增荧光检测方法的建立及应用

张紫薇¹^,²(), 黄春媛², 于娜¹^,³, 郑佳馨², 张艳², 刘光亮¹^,², 曹宗喜¹^,²^,³^,⁴()

1.新疆农业大学动物医学学院,乌鲁木齐 830052
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/国家动物疫病数据中心海口观测实验点,海南海口 571100
3.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
4.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:张紫薇(1999-),女,新疆沙湾人,硕士研究生,研究方向为家畜传染病学,(E-mail)1770735142@qq.com
基金资助:
国家自然科学基金项目(32060796);海南省重点研发项目(ZDYF2023XDNY038);国家重点研发计划项目(2023YFC3404302);国家重点研发计划项目(2021YFA0805905);海南省重大科技计划项目(ZDKJ2021030)

Abstract

Abstract:

【Objective】 There is a need for the development of a clinically rapid, simple, and accurate method for the detection of PRRSV. 【Methods】 This study aims to establish a method based on reverse transcription recombinase-mediated isothermal amplification fluorescence detection of PRRSV. The method was developed by designing specific primers and probes against the PRRSV ORF4 gene sequence. 【Results】 The fluorescent RT-RAA assay could be amplified with primers and probes at 41℃ for 20 min, and the results could be observed in real time by a fluorescence quantitative PCR instrument. The method had high sensitivity and strong specificity, and no cross-reactivity with other porcine pathogens. 【Conclusion】 The fluorescent RT-RAA and PCR used to test 180 samples showed the compliance rate between the two methods was 98.3%.

Key words: porcine reproductive and respiratory syndrome virus; ORF4; RAA; rapid detection

摘要：

【目的】开发研究一种临床上快速、简单和准确的检测PRRSV的方法。【方法】该研究建立一种基于逆转录重组酶介导等温扩增荧光检测PRRSV的方法。该方法通过针对PRRSV ORF4基因序列设计特异性引物和探针。【结果】荧光RT-RAA检测可在41℃下用引物和探扩增20 min,通过荧光定量PCR仪实时观察结果。该方法具有较高的灵敏度和较强的特异性,与其他猪病原体无交叉反应。【结论】使用荧光RT-RAA和PCR检测180份样品,两种方法之间的符合率98.3%。

关键词: 猪繁殖与呼吸综合征病毒, ORF4, RAA, 快速检测

CLC Number:

S828

ZHANG Ziwei, HUANG Chunyuan, YU Na, ZHENG Jiaxin, ZHANG Yan, LIU Guangliang, CAO Zongxi. Establishment and application of a fluorescent assay for reverse transcription recombinase-mediated isothermal amplification of porcine reproductive and respiratory syndrome virus[J]. Xinjiang Agricultural Sciences, 2025, 62(5): 1286-1292.

张紫薇, 黄春媛, 于娜, 郑佳馨, 张艳, 刘光亮, 曹宗喜. 猪繁殖与呼吸综合征病毒的逆转录重组酶介导等温扩增荧光检测方法的建立及应用[J]. 新疆农业科学, 2025, 62(5): 1286-1292.

Figures/Tables 7

Fig.1 Optimisation of probe concentration forPRRSV fluorescent RT-RAA methods Notes:1: probe final concentration of 0.1 μmol/L; 2: probe final concentration of 0.2 μmol/L; 3: probe final concentration of 0.3 μmol/L; 4: probe final concentration of 0.4 μmol/L; 5: negative control

Fig. 2 Optimisation of primer concentration for PRRSV fluorescent RT-RAA method Notes:1: primer final concentration of 0.3 μmol/L; 2: primer final concentration of 0.4 μmol/L; 3: primer final concentration of 0.5 μmol/L; 4: primer final concentration of 0.6 μmol/L; 5: negative control

Fig. 3 Optimisation of sample addition for the PRRSV fluorescence RT-RAA method Notes:1: 156 ng of total RNA; 2: 260 ng of total RNA; 3: 364 ng of total RNA; 4: 468 ng of total RNA; 5: negative control

Fig. 4 Optimisation of reaction temperature for PRRSV fluorescent RT-RAA method Notes:1: reaction temperature was 37°C; 2: reaction temperature was 39°C; 3: reaction temperature was 41°C; 4: reaction temperature was 43°C; 5: negative control

Fig.5 PRRSV fluorescent RT-RAA method sensitivity test Notes:1: 54 ng of total RNA; 2: 10.8 ng of total RNA; 3: 2.16 ng of total RNA; 4: 0.432 ng of total RNA; 5: 0.086,4 ng of total RNA; 6: negative control

Fig. 6 PRRSV fluorescent RT-RAA method specificity test results Notes:1: PRRSV NADC30 strain; 2: PRRSV TJM-F92 strain; 3: PRRSV R98 strain; 4-8: TGEV Purdue strain, CSFV C strain, PEDV LJX strain, PRV Bartha-K61 strain, negative control, respectively

Tab.1 Comparison of RT-RAA-LF and PCR methods for the detection of clinical samples

项目 Items		PCR		总量 Total (份)
项目 Items		阳性 Positive (份)	阴性 Negative (份)	总量 Total (份)
荧光RT-RAA 方法 Fluorescent RT-RAA method	阳性	73	3	76
	阴性	0	104	104
	总量	73	107	180

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Establishment and application of a fluorescent assay for reverse transcription recombinase-mediated isothermal amplification of porcine reproductive and respiratory syndrome virus

猪繁殖与呼吸综合征病毒的逆转录重组酶介导等温扩增荧光检测方法的建立及应用

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