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A multiplex digital PCR assay for detection and quantitation of porcine circovirus type 2 and type 3

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Abstract

Porcine circovirus (PCV) has become a major pathogen, causing major economic losses in the global pig industry, and PCV type 2 (PCV2) and 3 (PCV3) are distributed worldwide. We designed specific primer and probe sequences targeting PCV2 Cap and PCV3 Rap and developed a multiplex crystal digital PCR (cdPCR) method after optimizing the primer concentration, probe concentration, and annealing temperature. The multiplex cdPCR assay permits precise and differential detection of PCV2 and PCV3, with a limit of detection of 1.39 × 101 and 1.27 × 101 copies/reaction, respectively, and no cross-reaction with other porcine viruses was observed. The intra-assay and interassay coefficients of variation (CVs) were less than 8.75%, indicating good repeatability and reproducibility. To evaluate the practical value of this assay, 40 tissue samples and 70 feed samples were tested for both PCV2 and PCV3 by cdPCR and quantitative PCR (qPCR). Using multiplex cdPCR, the rates of PCV2 infection, PCV3 infection, and coinfection were 28.45%, 1.72%, and 12.93%, respectively, and using multiplex qPCR, they were 25.00%, 0.86%, and 4.31%, respectively This highly specific and sensitive multiplex cdPCR thus allows accurate simultaneous detection of PCV2 and PCV3, and it is particularly well suited for applications that require the detection of small amounts of input nucleic acid or samples with intensive processing and complex matrices.

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Data availability

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

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Funding

This work was supported by the National Key Research and Development Program of China (2021YFF0602800), the Key Research and Development Program of Zhejiang Province (2021C02060), and the Research Program of General Administration of Customs of China (2021HK59).

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Authors and Affiliations

  1. Zhejiang Academy of Science and Technology for Inspection and Quarantine, Hangzhou, 310016, China

    Jiangbing Shuai, Zhongcai Wang, Ruoxue Zeng & Xiaofeng Zhang

  2. College of Animal Science and Technology, Zhejiang Agriculture & Forestry University, Hangzhou, 311300, China

    Kexin Chen & Houhui Song

  3. Zhejiang Provincial Key Laboratory of Biometrology and Inspection & Quarantine, China Jiliang University, Hangzhou, 310018, China

    Biao Ma & Mingzhou Zhang

Authors
  1. Jiangbing Shuai
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  2. Kexin Chen
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  5. Biao Ma
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  6. Mingzhou Zhang
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  7. Houhui Song
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  8. Xiaofeng Zhang
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Contributions

All authors contributed to the study conception and design. Material preparation, data collection, and analysis: Jiangbing Shuai and Kexin Chen. Conceptualization: Jiangbing Shuai, Houhui Song, and Xiaofeng Zhang. Formal analysis: Kexin Chen, Zhongcai Wang, and Ruoxue Zeng. Preliminary inspection, of samples: Biao Ma and Mingzhou Zhang. The first draft of the manuscript was written by Jiangbing Shuai and Kexin Chen, and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Xiaofeng Zhang.

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Shuai, J., Chen, K., Wang, Z. et al. A multiplex digital PCR assay for detection and quantitation of porcine circovirus type 2 and type 3. Arch Virol 169, 119 (2024). https://doi.org/10.1007/s00705-024-06044-0

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