Skip to main content

Advertisement

SpringerLink
Log in

Development and use of a droplet digital PCR (ddPCR) assay to achieve sensitive and fast atypical porcine pestivirus detection

  • Bacterial, Fungal and Virus Molecular Biology - Research Paper
  • Published:
Brazilian Journal of Microbiology Aims and scope Submit manuscript

Abstract

Atypical porcine pestivirus (APPV) is a recently discovered RNA virus, which mainly caused congenital tremor in piglets. Droplet digital PCR (ddPCR) is an absolute quantitative method that does not rely on the standard curve but has high sensitivity and accuracy. The present study aimed to develop a ddPCR detection assay for APPV. Furthermore, we evaluated the limit of detection, sensitivity, specificity and reproducibility of the ddPCR and real-time quantitative PCR (qPCR) and tested 135 clinical samples to calculate the detection rate of the two methods. The results showed that both methods had a strong linear relationship and quantitative correlation. The ddPCR assay had a minimum detection limit of 0.15 copies/μL for APPV, with a sensitivity 100 times that of qPCR. We tested clinical samples and found that the APPV ddPCR had a 27.4% positive detection rate, noticeably higher than that of the qPCR (14.8%). Additionally, the APPV ddPCR method had excellent repeatability and specificity. In brief, our study provided a novel, feasible and sensitive diagnostic technique to identify and monitor APPV.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

References

  1. Done JT, Woolley J, Upcott DH, Hebert CN (1986) Porcine congenital tremor type AII: Spinal cord morphometry. Br Vet J 142(2):0–150. https://doi.org/10.1016/0007-1935(86)90090-4

    Article  CAS  Google Scholar 

  2. Blakemore WF, Harding JD, Done JT (1974) Ultrastructural Observations on the Spinal Cord of a Landrace Pig with Congenital Tremor Type AIII. Res Vet Sci 17(2):174–178. https://doi.org/10.1016/S0034-5288(18)33677-4

    Article  CAS  PubMed  Google Scholar 

  3. Patterson DS, Sweasey D, Brush PJ, Harding JD (1973) Neurochemistry of the spinal cord in British Saddleback piglets affected with congenital tremor, type A-IV, a second form of hereditary cerebrospinal hypomyelinogenesis. J Neurochem 21:397–406. https://doi.org/10.1111/j.1471-4159.1973.tb04259.x

    Article  CAS  PubMed  Google Scholar 

  4. Knox B, Askaa J, Basse A, Bitsch V, Eskildsen M, Mandrup M, Ottosen HE, Overby E, Pedersen KB, Rasmussen F (1980) Congenital ataxia and tremor with cerebellar hypoplasia in piglets borne by sows treated with Neguvon vet. (metrifonate, trichlorfon) during pregnancy. Nord Vet Med 30:538–545. https://doi.org/10.1080/00480169.1978.34583

    Article  Google Scholar 

  5. Hause BM, Collin EA, Peddireddi L, Yuan F, Chen Z, Hesse RA, Gauger PC, Clement T, Fang Y, Anderson G (2015) Discovery of a novel putative atypical porcine pestivirus in pigs in the USA. J Gen Virol 96(10):2994. https://doi.org/10.1099/jgv.0.000251

    Article  CAS  PubMed  Google Scholar 

  6. Postel A, Hansmann F, Baechlein C, Fischer N, Alawi M, Grundhoff A, Derking S, Tenhündfeld J, Pfankuche VM, Herder V, Baumgärtner W, Wendt M, Becher P (2016) Presence of atypical porcine pestivirus (APPV) genomes in newborn piglets correlates with congenital tremor. Sci Rep 6:27735. https://doi.org/10.1038/srep27735

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Schwarz L, Riedel C, Högler S, Sinn LJ, Voglmayr T, Wöchtl B, Dinhopl N, Rebel BB, Weissenböck H, Ladinig A, Rümenapf T, Lamp B (2017) Congenital infection with atypical porcine pestivirus (APPV) is associated with disease and viral persistence. Vet Res 48(1):1. https://doi.org/10.1186/s13567-016-0406-1

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Yang XY, Chen SJ, Lin H, Zhang Q, An H, Zhu L (2020) Establishment and application of multiplex TaqMan fluorescence quantitative PCR for detection of congenital tremor-related viruses in piglets (in Chinese). Jiangsu J Agric Sci 36(4):971–977

    Google Scholar 

  9. Yuan J, Han Z, Li J, Huang Y, Yang J, Ding H, Zhang J, Zhu M, Zhang Y, Liao J, Zhao M, Chen J (2017) Atypical Porcine Pestivirus as a Novel Type of Pestivirus in Pigs in China. Front Microbiol 8:862. https://doi.org/10.3389/fmicb.2017.00862

    Article  PubMed  PubMed Central  Google Scholar 

  10. Gatto IRH, Arruda PH, Visek CA, Victoria JG, Patterson AR, Krull AC, Schwartz KJ, de Oliveira LG, Arruda BL (2018) Detection of atypical porcine pestivirus in semen from commercial boar studs in the United States. Transbound Emerg Dis 65(2):e339–e343. https://doi.org/10.1111/tbed.12759

    Article  CAS  PubMed  Google Scholar 

  11. de Groof A, Deijs M, Guelen L, van Grinsven L, van Os-Galdos L, Vogels W, Derks C, Cruijsen T, Geurts V, Vrijenhoek M, Suijskens J, van Doorn P, van Leengoed L, Schrier C, van der Hoek (2016) Atypical Porcine Pestivirus: A Possible Cause of Congenital Tremor Type A-II in Newborn Piglets. Viruses 8(10):271. https://doi.org/10.3390/v8100271

  12. Postel A, Meyer D, Cagatay GN, Feliziani F, De Mia GM, Fischer N, Grundhoff A, Milićević V, Deng MC, Chang CY, Qiu HJ, Sun Y, Wendt M, Becher P (2017) High Abundance and Genetic Variability of Atypical Porcine Pestivirus in Pigs from Europe and Asia. Emerg Infect Dis 23(12):2104–2107. https://doi.org/10.3201/eid2312.170951

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Bauermann FV, Ridpath JF (2015) HoBi-like viruses-the typical “atypical bovine pestivirus”. Anim Health Res Rev 16(1):64–69. https://doi.org/10.1017/S146625231500002X

    Article  PubMed  Google Scholar 

  14. Ryan MD, Monaghan S, Flint M (1998) Virus-encoded proteinases of the Flaviviridae. J Gen Virol 5:947–959. https://doi.org/10.1099/0022-1317-79-5-947

    Article  Google Scholar 

  15. Dingle TC, Sedlak RH, Cook L, Jerome KR (2013) Tolerance of Droplet-Digital PCR vs Real-Time Quantitative PCR to Inhibitory Substances. Clin Chem 59(11):1670–1672. https://doi.org/10.1373/clinchem.2013.211045

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Vogelstein B, Kinzler KW (1999) Digital PCR. Proc Natl Acad Sci USA 96(16):9236–9241. https://doi.org/10.1073/pnas.96.16.9236

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Sanders R, Huggett JF, Bushell CA, Cowen S, Scott DJ, Foy CA (2011) Evaluation of digital PCR for absolute DNA quantification. Anal Chcm 83:6417–6484. https://doi.org/10.1021/ac103230c

    Article  CAS  Google Scholar 

  18. Pinheiro LB, Coleman VA, Hindson CM, Herrmann J, Hindson BJ, Bhat S, Emslie KR (2012) Evaluation of a droplet digital polymerase chain reaction format for DNA copy number quantification. Anal Chem 84:1003–1011. https://doi.org/10.1021/ac202578x

    Article  CAS  PubMed  Google Scholar 

  19. White RA, Quake SR, Curr K (2012) Digital PCR provides absolute quantitation of viral load for an occult RNA virus. J Virol Methods 179:15–50. https://doi.org/10.1016/j.jviromet.2011.09.017

    Article  CAS  Google Scholar 

  20. Miotto E, Saccenti E, Lupini L, Callegari E, Negrini M, Ferracin M (2014) Quantification of circulating miRNAs by ddPCR: comparison of EvaGreen-and TaqMan-based chemistries. Cancer Epidemiol Biomarkers Prev 23(12):2638–2642. https://doi.org/10.1158/1055-9965.EPI-14-0503

    Article  CAS  PubMed  Google Scholar 

  21. Ling Ling, Michael S, Rachel C, Damien B, John W, Howard S (2015) A powerful new approach to measuring engraftment using copy number variations and ddPCR, exemplified in a complex allogeneic bone marrow transplantation case. Pathology 47:S87. https://doi.org/10.1097/01.PAT.0000461574.20652.e5

  22. Zhang Y, Tang ET, Du Z (2016) Detection of MET Gene Copy Number in Cancer Samples Using the ddPCR Method. PLoS ONE 11(1):e0146784. https://doi.org/10.1371/journal.pone.0146784

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Bharuthram A, Paximadis M, Picton AC, Tiemessen CT (2014) Comparison of a quantitative real-time RT-PCR assay and ddPCR for copy number analysis of the CCL4L genes. Infect Genet Evol 25:28–35. https://doi.org/10.1016/j.meegid.2014.03.028

    Article  CAS  PubMed  Google Scholar 

Download references

Funding

This research was supported by the Sichuan Province's “14th Five-Year Plan” Sichuan pig major science and technology project (Project No: 2021ZDZX0010), the R&D key research and development project of Sichuan science and technology plan (Project No: 2020YFN0147) and the Sichuan veterinary medicine and drug innovation group of the China agricultural research system (Project No: CARS-SVDIP).

Author information

Author notes

  1. Lishuang Deng and Xiaoyu Yang contributed equally to this work.

Authors and Affiliations

  1. College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, 611130, China

    Lishuang Deng, Xiaoyu Yang, Zhiwen Xu, Fengqin Li, Jun Zhao, Huidan Deng, Zhijie Jian, Xiangang Sun & Ling Zhu

  2. Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu, 611130, China

    Zhiwen Xu & Ling Zhu

Authors
  1. Lishuang Deng
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Xiaoyu Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Zhiwen Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Fengqin Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jun Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Huidan Deng
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Zhijie Jian
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Xiangang Sun
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Ling Zhu
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ling Zhu.

Ethics declarations

Conflict of interest

The authors declare no competing interests.

Additional information

Responsible Editor: Flavio Guimaraes Fonseca

Publisher's note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Lishuang Deng and Xiaoyu Yang share co-first authorship.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 34 KB)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Deng, L., Yang, X., Xu, Z. et al. Development and use of a droplet digital PCR (ddPCR) assay to achieve sensitive and fast atypical porcine pestivirus detection. Braz J Microbiol 53, 625–631 (2022). https://doi.org/10.1007/s42770-022-00728-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s42770-022-00728-y

Keywords

Search

Navigation