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Bead-based suspension array for simultaneous differential detection of five major swine viruses

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Abstract

A novel multiplex detection array based on Luminex xMAP technology was developed and validated for simultaneous detection of five major viruses causing swine reproductive diseases. By combining one-step asymmetric multiplex reverse transcription polymerase chain reaction (RT-PCR) with xMAP bead-based hybridization and flow cytometry analysis, the resulting multiplex assay was capable of detecting single and mixed infections of PRRSV, PCV-2, PRV, CSFV, and PPV in a single reaction. The assay accurately detected and differentiated 23 viral strains used in this study. The low detection limit was determined as 2.2–22 copies/μL (corresponding to 0.5–6.8 fg/μL DNA template) on plasmid constructs containing viral fragments. The intra-assay and inter-assay variances (CV%) were low that ranged from 2.5 to 5.4 % and 4.1 to 7.6 %, respectively. The assay was applied to test field samples and detected single and mixed viral infections. The detection rate was higher than that of uniplex conventional PCR and RT-PCR methods. The detection of PRRSV by the bead-based multiplex assay was comparable with a commercially available real time RT-PCR kit. The test procedure on purified DNA or RNA samples could be completed within 2 h. In conclusion, the bead-based suspension array presented here proved to be a high-throughput practical tool that provided highly specific and sensitive identification of single and multiple infections of five major viruses in pigs and boar semen.

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Acknowledgments

We thank Ms. Yan-Yu Duan and Ms. Zhi-Ling Liu for technical assistance. This work was supported by the research program (No. 2013IK029) of General Administration of Quality Supervision, Inspection and Quarantine of the People’s Republic of China (AQSIQ), and the Science and Technology Research Program (No. 2012B020305003) of Guangdong province, China.

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Authors and Affiliations

  1. Animal Inspection and Quarantine Laboratory, Technical Center, Guangdong Entry–Exit Inspection and Quarantine Bureau, No.66 Huacheng Dadao Ave., Zhujiangxincheng, Guangzhou, 510623, China

    Ru Chen

  2. School of Life Sciences, Sun Yat-Sen University, Guangzhou, China

    Xiao-Lu Yu, Cun-Yi Xue & Yong-Chang Cao

  3. Department of Genetics, National Research Institute for Family Planning, Beijing, 100081, China

    Xiao-Bo Gao

  4. Veterinary Medicine Institute, Guangdong Academy of Agricultural Sciences, Guangzhou, China

    Chang-Xu Song & Yan Li

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  1. Ru Chen
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  2. Xiao-Lu Yu
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  3. Xiao-Bo Gao
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  4. Cun-Yi Xue
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  5. Chang-Xu Song
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  6. Yan Li
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  7. Yong-Chang Cao
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Correspondence to Ru Chen.

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Chen, R., Yu, XL., Gao, XB. et al. Bead-based suspension array for simultaneous differential detection of five major swine viruses. Appl Microbiol Biotechnol 99, 919–928 (2015). https://doi.org/10.1007/s00253-014-6337-8

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  • DOI: https://doi.org/10.1007/s00253-014-6337-8

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