Korean Journal of Veterinary Service (한국동물위생학회지)

The Korean Society of Veterinary Service (한국동물위생학회)
권호 표지
DOI QR코드

DOI QR Code

Development of a real-time polymerase chain reaction assay for reliable detection of a novel porcine circovirus 4 with an endogenous internal positive control

  • Kim, Hye-Ryung (College of Veterinary Medicine & Animal Disease Intervention Center, Kyungpook National University) ;
  • Park, Jonghyun (College of Veterinary Medicine & Animal Disease Intervention Center, Kyungpook National University) ;
  • Park, Ji-Hoon (College of Veterinary Medicine & Animal Disease Intervention Center, Kyungpook National University) ;
  • Kim, Jong-Min (College of Veterinary Medicine & Animal Disease Intervention Center, Kyungpook National University) ;
  • Baek, Ji-Su (College of Veterinary Medicine & Animal Disease Intervention Center, Kyungpook National University) ;
  • Kim, Da-Young (Foreign Animal Disease Division, Animal and Plant Quarantine Agency (APQA)) ;
  • Lyoo, Young S. (College of Veterinary Medicine, Konkuk University) ;
  • Park, Choi-Kyu (College of Veterinary Medicine & Animal Disease Intervention Center, Kyungpook National University)
  • Received : 2022.03.26
  • Accepted : 2022.03.29
  • Published : 2022.03.30
Download PDF
⟨ Previous Next ⟩

Abstract

A novel porcine circovirus 4 (PCV4) was recently identified in Chinese and Korean pig herds. Although several conventional polymerase chain reaction (cPCR) and real-time PCR (qPCR) assays were used for PCV4 detection, more sensitive and reliable qPCR assay is needed that can simultaneously detect PCV4 and internal positive control (IPC) to avoid false-negative results. In the present study, a duplex qPCR (dqPCR) assay was developed using primers/probe sets targeting the PCV4 Cap gene and pig (glyceraldehyde-3-phosphate dehydrogenase) GAPDH gene as an IPC. The developed dqPCR assay was specifically detected PCV4 but not other PCVs and porcine pathogens, indicating that the newly designed primers/probe set is specific to the PCV4 Cap gene. Furthermore, GAPDH was stably amplified by the dqPCR in all tested viral and clinical samples containing pig cellular materials, indicating the high reliability of the dqPCR assay. The limit of detection of the assay 5 copies of the target PCV4 genes, but the sensitivity of the assay was higher than that of the previously described assays. The assay demonstrated high repeatability and reproducibility, with coefficients of intra-assay and inter-assay variation of less than 1.0%. Clinical evaluation using 102 diseased pig samples from 18 pig farms showed that PCV4 circulated in the Korean pig population. The detection rate of PCV4 obtained using the newly developed dqPCR was 26.5% (27/102), which was higher than that obtained using the previously described cPCR and TaqMan probe-based qPCR and similar to that obtained using the previously described SYBR Green-based qPCR. The dqPCR assay with IPC is highly specific, sensitive, and reliable for detecting PCV4 from clinical samples, and it will be useful for etiological diagnosis, epidemiological study, and control of the PCV4 infections.

Keywords

Acknowledgement

This research was supported by the Commercializations Promotion Agency for R&D Outcomes (COMPA) grant funded by the Korean Government (Ministry of Science and ICT) (R&D project No. 1711139487), Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry (IPET) through "Animal Disease Management Technology Development Program (321015-01-1-CG000)", and "Cooperative Research Program for Agriculture Science and Technology Development (Project No. PJ01561102)" funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA), Rural Development Administration (RDA), Republic of Korea.

References

  1. Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl MW, Shipley GL, Handsomely J, WittwerCT. 2009. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem. 55: 611-622. https://doi.org/10.1373/clinchem.2008.112797
  2. Chen N, Xiao Y, Li X, Li S, Xie N, Yan X, Li X, Zhu J. 2021. Development and application of a quadruplex real-time PCR assay for differential detection of porcine circoviruses (PCV1 to PCV4) in Jiangsu province of China from 2016 to 2020. Transbound Emerg Dis 68: 1615-1624. https://doi.org/10.1111/tbed.13833
  3. Duvigneau JC, Hartl RT, Groiss S & Gemeiner M. 2005. Quantitative simultaneous multiplex real-time PCR for the detection of porcine cytokines. J Immunol methods 306: 16-27. https://doi.org/10.1016/j.jim.2005.06.021
  4. Ha Z, Yu C, Xie C, Wang G, Zhang Y, Hao P, Li J, Li Z, Li Y, Rong F, Nan F, Zhang H, Zhuang X, Xie Y, Shi N, Lu H, Jin N. 2021. Retrospective surveillance of porcine circovirus 4 in pigs in Inner Mongolia, China, from 2016 to 2018. Arch Virol 166: 1951-1959. https://doi.org/10.1007/s00705-021-05088-w
  5. Hou CY, Xu T, Zhang LH, Cui JT, Zhang YH, Li XS, Zheng LL, Chen HY. 2021. Simultaneous detection and differentiation of porcine circovirus 3 and 4 using a SYBR Green I-based duplex quantitative PCR assay. J Virol Methods 293: 114152. https://doi.org/10.1016/j.jviromet.2021.114152
  6. Kim DY, Kim HR, Park JH, Kwon NY, Kim JM, Kim JK, Park JH, Lee KK, Kim SH, Kim WI, Lyoo YS, Park CK. 2022. Detection of a novel porcine circovirus 4 in Korean pig herds using a loop-mediated isothermal amplification assay. J Virol Methods 299: 114350. https://doi.org/10.1016/j.jviromet.2021.114350
  7. Kim HR., Park YR., Lim DR, Park MJ, Park JY, Kim SH, Lee KK, Lyoo YS, Park CK. 2017. Multiplex realtime polymerase chain reaction for the differential detection of porcine circovirus 2 and 3. J Virol Methods 250: 11-16. https://doi.org/10.1016/j.jviromet.2017.09.021
  8. Kwiecien R, Kopp-Schneider A, Blettner M. 2011. Concordance analysis: part 16 of a series on evaluation of scientific publications. Deutsches Arzteblatt International 108: 515.
  9. Navarro E, Serrano-Heras G, Castanoa MJ, Solera J. 2015. Real-time PCR detection chemistry. Clinica Chimica Acta 439: 231-250. https://doi.org/10.1016/j.cca.2014.10.017
  10. Nguyen VG, Do HQ, Huynh TM, Park YH, Park BK, Chung HC. 2021. Molecular-based detection, genetic characterization and phylogenetic analysis of porcine circovirus 4 from Korean domestic swine farms. Transbound Emerg Dis 00: 1-11.
  11. Park CK, Lee KK, Kim HS. 2004. Genetic characterization of porcine circovirus 2 Korean isolates. Korean J Vet Res 44: 571-579.
  12. Rosario K, Breitbart M, Harrach B, Segales J, Delwart E, Biagini P, Varsani A. 2017.Revisiting the taxonomy of the family Circoviridae: establishment of the genus Cyclovirus and removal of the genus Gyrovirus. Arch Virol 162: 1447-1463. https://doi.org/10.1007/s00705-017-3247-y
  13. Sun W, Du Q, Han Z, Bi J, Lan T, Wang W, Zheng M. 2021. Detection and genetic characterization of porcine circovirus 4 (PCV4) in Guangxi, China. Gene 773: 145384. https://doi.org/10.1016/j.gene.2020.145384
  14. Tajadini M, Panjehpour M, Javanmard SH. 2014. Comparison of SYBR Green and TaqMan methods in quantitative real-time polymerase chain reaction analysis of four adenosine receptor subtypes. Adv Biomed Res 3: 85. https://doi.org/10.4103/2277-9175.127998
  15. Tian RB, Zhao Y, Cui JT, Xheng HH, Xu T, Hou CY, Wang ZY, Li XS, Zheng LL, Chen HY. 2021. Molecular detection and phylogenetic analysis of porcine circovirus 4 in Henan and Shanxi Provinces of China. Transbound Emerg Dis 68: 276-282. https://doi.org/10.1111/tbed.13714
  16. Zhang D, Bai C, Ge K, Li Y, Gao W, Jiang S, Wang Y. 2020a. Establishment of an SYBR Green-based real-time PCR assay for porcine circovirus type 4 detection. J. Virol Methods 285: 113963. https://doi.org/10.1016/j.jviromet.2020.113963
  17. Zhang HH, Hu WQ, Li JY, Liu TN, Zhou JY, Opriessnig T, Xiao CT. 2020b. Novel circovirus species identified in farmed pigs designated as porcine circovirus 4, Hunan province, China. Transbound Emerg Dis 67: 1057-1061. https://doi.org/10.1111/tbed.13446