Please wait a minute...
Journal of Integrative Agriculture
Advanced Online Publication | Current Issue | Archive | Adv Search
A one-pot RAA-CRISPR/Cas12a assay for the visual detection of Pseudorabies virus

Boyi Li1*, Junjie Zhao1, 2*, Luyao Liu1, Ziyang Han1, Zimo Zhang1, Yuxin Zhang1, Qinghui Qi1, Hualei Wang1, Hongli Jin3, Pei Huang1, Haili Zhang1#

1 State Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases/Key Laboratory for Zoonosis Research of the Ministry of Education, Institute of Zoonosis, College of Veterinary Medicine, Jilin University, Jilin 130062, China

2 National/WOAH Reference Laboratory for Classical Swine Fever, China Institute of Veterinary Drug Control, Beijing 100081, China

3 Changchun SR Biological Technology Co., LTD, Changchun 130012, China

 Highlights: 

We developed a nucleic acid detection method for pseudorabies virus (PRV) by integrating recombinase-aided amplification (RAA) with the CRISPR/Cas12a system. The assay achieved a limit of detection (LOD) of 5×100 copies μL-1 for recombinant plasmid and 7.96×10-2 TCID50 per reaction for the virus within 50 minutes. This method provides strong support for the clinical diagnosis and field surveillance of PRV.

Download:  PDF in ScienceDirect  
Export:  BibTeX | EndNote (RIS)      
摘要  

猪伪狂犬病(Pseudorabies, PR)是由伪狂犬病病毒(Pseudorabies virus, PRV)引起的一种重要动物传染病,不仅给全球养猪业造成重大经济损失,还存在感染人并引发脑炎的人兽共患风险。近年来,新型PRV变异株的出现降低了传统疫苗的保护效果,因此建立快速、简便的现场检测方法对防控该病具有重要意义。为此,本研究将重组酶辅助扩增(RAA)与CRISPR/Cas12a系统相结合,针对PRVgE基因设计特异性引物与crRNA,建立了一管法核酸检测体系,并优化了反应条件,进而评价了该方法的灵敏度、特异性及临床样本检测一致性。结果显示,所建立的RAA-CRISPR/Cas12a方法可在50分钟内完成检测,对重组质粒的检测限为5拷贝/μL,对PRV病毒的检测限为7.96×10-2 TCID50/反应;该方法与猪瘟病毒(CSFV)、非洲猪瘟病毒(ASFV)、猪丁型冠状病毒(PDCoV)等其他常见猪病原体均无交叉反应,表现出高度特异性;在临床样本验证中,本方法与实时定量聚合酶链式反应(qPCR)检测结果完全一致,具有良好的临床符合性。综上所述,本研究成功建立了一种基于RAACRISPR/Cas12a系统的PRV核酸检测方法,具有快速、灵敏、特异、操作简便等优点,适用于资源有限条件下的现场检测,为PR的临床诊断提供了有力工具。



Online: 25 April 2026  
Fund: 

This work was financially supported by the National Key Research and Development Program of China (grant No.2021YFF0703600) and Jilin Provincial Department of Science and Technology Project (grant No.20250202061NC).

About author:  Boyi Li, E-mail: liboyi1029@163.com; Junjie Zhao, E-mail: 1229195209@qq.com; #Correspondence: Haili Zhang, E-mail: zhanghaili@jlu.edu.cn * These authors contributed equally to this work.

Cite this article: 

Boyi Li, Junjie Zhao, Luyao Liu, Ziyang Han, Zimo Zhang, Yuxin Zhang, Qinghui Qi, Hualei Wang, Hongli Jin, Pei Huang, Haili Zhang. 2026. A one-pot RAA-CRISPR/Cas12a assay for the visual detection of Pseudorabies virus. Journal of Integrative Agriculture, Doi:10.1016/j.jia.2026.04.027

Cao Z, Li Z, Jin K, Huang Z, Hu R, Shi J, Yu H, Wang G, He W, Wang H, Lan Y, Zhang H. 2025. A rapid and visual detection method for Porcine deltacoronavirus with single-copy sensitivity based on the CRISPR/Cas12a assay. Journal of Integrative Agriculture, 25, 1736-1739.

Chen J, Li G, Wan C, Li Y, Peng L, Fang R, Peng Y, Ye C. 2022. A comparison of Pseudorabies virus latency to other α-Herpesvirinae subfamily members. Viruses, 14, doi: 10.3390/v1407138.

Chen J, Zhao G, Yang Y, Li Y, Song Y, Li D, Du Q, Tong D, Huang Y. 2025a. Pseudorabies virus induces natural killer cell depletion by GSDMD-mediated inflammation and pyroptosis to promote infection and lung injury. Journal of Virology, 99, doi: 10.1128/jvi.00415-25.

Chen Y, Gao J, Hua R, Zhang G. 2025b. Pseudorabies virus as a zoonosis: scientific and public health implications. Virus Genes, 61, 9-25.

Fan S, Yuan H, Liu L, Li H, Wang S, Zhao W, Wu Y, Wang P, Hu Y, Han J, Lyu Y, Zhang W, Chen P, Wu H, Gong Y, Ma Z, Li Y, Yu J, Qiao X, Li G, Zhao Y, Wang D, Ren H, Peng B, Cui L, Wang J, Guan H. 2020. Pseudorabies virus encephalitis in humans: A case series study. Journal of NeuroVirology, 26, 556-564.

Hernández F A, Carr A N, Milleson M P, Merrill H R, Avery M L, Parker B M, Pylant C L, Austin J D, Wisely S M. 2020. Dispersal and land cover contribute to Pseudorabies virus exposure in invasive wild pigs. Ecohealth, 17, 498-511.

Homchan A, Patchsung M, Chantanakool P, Wongsatit T, Onchan W, Muengsaen D, Thaweeskulchai T, Tandean M, Sakpetch T, Suraritdechachai S, Aphicho K, Panchai C, Taiwan S, Horthongkham N, Sudyoadsuk T, Reinhardt A, Uttamapinant C. 2025. Recombinase-controlled multiphase condensates accelerate nucleic acid amplification and CRISPR-based diagnostics. Journal of the American Chemical Society, 147, 10088-10103.

Jiang S, Li H, Zhang L, Mu W, Zhang Y, Chen T, Wu J, Tang H, Zheng S, Liu Y, Wu Y, Luo X, Xie Y, Ren J. 2025. Generic Diagramming Platform (GDP): A comprehensive database of high-quality biomedical graphics. Nucleic Acids Research, 53, D1670-d1676.

Li J, Wang Y, Wang B, Lou J, Ni P, Jin Y, Chen S, Duan G, Zhang R. 2022. Application of CRISPR/Cas systems in the nucleic acid detection of infectious diseases. Diagnostics (Basel), 12, doi: 10.3390/diagnostics12102455.

Li X, Zhu S, Zhang X, Ren Y, He J, Zhou J, Yin L, Wang G, Zhong T, Wang L, Xiao Y, Zhu C, Yin C, Yu X. 2023. Advances in the application of recombinase-aided amplification combined with CRISPR-Cas technology in quick detection of pathogenic microbes. Front Bioeng Biotechnol, 11, doi: 10.3389/fbioe.2023.1215466.

Liu A, Xue T, Zhao X, Zou J, Pu H, Hu X, Tian Z. 2022a. Pseudorabies virus associations in wild animals: Review of potential reservoirs for cross-host transmission. Viruses, 14, doi: 10.3390/v14102254.

Liu Q, Kuang Y, Li Y, Guo H, Zhou C, Guo S, Tan C, Wu B, Chen H, Wang X. 2022b. The epidemiology and variation in Pseudorabies virus: A continuing challenge to pigs and humans. Viruses, 14, doi: 10.3390/v14071463.

Ma Y, Shi K, Chen Z, Shi Y, Zhou Q, Mo S, Wei H, Hu L, Mo M. 2024. Simultaneous detection of Porcine respiratory coronavirus, Porcine reproductive and respiratory syndrome virus, Swine influenza virus, and Pseudorabies virus via Quadruplex One-Step RT-qPCR. Pathogens, 13, doi: 10.3390/pathogens13040341.

Sehl J, Teifke J P. 2020. Comparative pathology of Pseudorabies in different naturally and experimentally infected species-A review. Pathogens, 9, doi: 10.3390/pathogens9080633.

Tan L, Zhu P, Getu Z, Yang X, Zheng S, Duan Y, Wang J, Zhou Y, Hu Y, Wang Y, Yang Y, Zuo M, Yao J. 2025. Antiviral activity of nitazoxanide against Pseudorabies virus infection in vitro. Frontiers In Veterinary Science, 12, doi: 10.3389/fvets.2025.1623545.

Tian X, Wang H, Song H, Wei Z, Zhu X, Liu G, Sun M, Huang X, Chen M, Tang Y, Wang H, Yang Y, An T. 2025. Recombinant Pseudorabies virus expressing the consensus VP2 protein of Porcine parvovirus 1 (PPV1) protects pigs against Pseudorabies virus and PPV1. Veterinary Research, 56, doi: 10.1186/s13567-025-01592-y.

Tu F, Zhang Y, Xu S, Yang X, Zhou L, Ge X, Han J, Guo X, Yang H. 2022. Detection of Pseudorabies virus with a real-time recombinase-aided amplification assay. Transboundary And Emerging Diseases, 69, 2266-2274.

Wang T Y, Li C, Cai X H, Shan T, Tang Y D. 2026. Spillover of Pseudorabies virus variants to humans: An urgent call for Pseudorabies eradication in domestic pigs. The Lancet Microbe, doi: 10.1016/j.lanmic.2026.101351.

WOAH. Codes and manuals. https://www.woah.org/en/what-we-do/standards/codes-and-manuals/

Zheng H H, Fu P F, Chen H Y, Wang Z Y. 2022. Pseudorabies virus: From pathogenesis to prevention strategies. Viruses, 14, doi: 10.3390/v14081638.

Zhuang L, Gong J, Shen J, Zhao Y, Yang J, Liu Q, Zhang Y, Shen Q. 2025. Advances in molecular epidemiology and detection methods of Pseudorabies virus. Discover Nano, 20, doi: 10.1186/s11671-025-04217-7.

No related articles found!
No Suggested Reading articles found!