Acta Veterinaria et Zootechnica Sinica ›› 2024, Vol. 55 ›› Issue (1): 413-418.doi: 10.11843/j.issn.0366-6964.2024.01.040

• RESEARCH NOTES • Previous Articles    

Establishment and Preliminary Application of a Quantitative Droplet Digital PCR Assay for Porcine Epidemic Diarrhea Virus

ZHOU Jianhao1, WANG Dongfang2, LIU Ying2, WANG Shujuan2, MA Zhenyuan2, XIE Caihua2, ZHAO Xueli2, YANG Haibo2, FENG Guidan4, KANG Taisheng3, HU Yufeng1, LI Bowen3, YAN Ruoqian2*   

  1. 1. College of Veterinary Medicine, Henan Agricultural University, Zhengzhou 450046, China;
    2. Henan Centre for Animal Diseases Control and Prevention/Henan Provincial Key Laboratory of Monitoring, Early Warning and Prevention and Control of Major Animal Diseases, Zhengzhou 450008, China;
    3. College of Animal Science and Technology, Henan University of Science and Technology, Luoyang 471000, China;
    4. Shanghai Animal Disease Prevention and Control Center, Shanghai 201103, China
  • Received:2023-04-11 Online:2024-01-23 Published:2024-01-24

PDF (PC)

102

Abstract: The aim of this study was to develop a highly sensitive, specific and reproducible method for the quantitative detection of porcine epidemic diarrhea virus (PEDV) by droplet digital PCR (ddPCR). In this study, we designed and synthesized specific primer pairs and probes based on the conserved region of the M gene sequence of PEDV (MK862249.1) registered in GenBank, and successfully established a fluorescent quantitative real-time PCR (FQ-PCR) for the detection of PEDV by optimizing the reaction system and reaction conditions. By optimizing the reaction system and reaction conditions, a fluorescent quantitative real-time PCR (FQ-PCR) method for PEDV detection was successfully established, and the primer pairs/probes of the self-established FQ-PCR method were used to determine the ddPCR method; sensitivity, specificity, reproducibility and preliminary application tests of the ddPCR method were performed. The results showed that the self-established FQ-PCR method outperformed the industrial standard FQ-PCR method (SN/T 1699—2017) in terms of sensitivity and reproducibility; the lowest detection limit of the ddPCR method based on the self-designed FQ-PCR method was 0.15 copies·μL-1, which was more sensitive than the industrial standard FQ-PCR (1.0×101 copies·μL-1); The linearity was good (R2>0.99) at template concentrations of 1.0×100 to 1.0×104 copies·μL-1. The intra- and inter-batch coefficients of variation (CV%) ranged from 1.52% to 7.40%. The results were negative for 11 control viruses including porcine delta coronavirus and porcine pseudorabies virus. The PEDV nucleic acid test was performed on 150 clinical samples by the established ddPCR and FQ-PCR methods, and the coincidence rate of the ddPCR method with the FQ-PCR method in detecting the positive samples was 100%.The PEDV ddPCR assay successfully established in this study can be used for early detection and quantitative detection of PEDV infection in clinical settings, and provides a quantitative means for the development of PEDV nucleic acid standards.

Key words: porcine epidemic diarrhea virus, droplet, digital PCR, method, establishment

CLC Number: