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. 2019 Nov;66(6):2271-2278.
doi: 10.1111/tbed.13276. Epub 2019 Jul 7.

Development of universal and quadruplex real-time RT-PCR assays for simultaneous detection and differentiation of porcine reproductive and respiratory syndrome viruses

Nanhua Chen  1 , Mengxue Ye  1 , Yanzhao Xiao  1 , Shuai Li  1 , Yucheng Huang  1 , Xiangdong Li  2 , Kegong Tian  2   3   4 , Jianzhong Zhu  1
Affiliations

Development of universal and quadruplex real-time RT-PCR assays for simultaneous detection and differentiation of porcine reproductive and respiratory syndrome viruses

Nanhua Chen et al. Transbound Emerg Dis. 2019 Nov.

Abstract

Porcine reproductive and respiratory syndrome virus 1 (PRRSV1) and 2 (PRRSV2) (including 3 major subtypes: classical (CA-PRRSV2), highly pathogenic (HP-PRRSV2) and NADC30-like (NL-PRRSV2)) are currently coexisting in Chinese swine herds but with distinct virulence. Reliable detection and differentiation assays are crucial to monitor the prevalence of PRRSV and to adopt effective control strategies. However, current diagnostic methods cannot simultaneously differentiate the four major groups of PRRSV in China. In this study, universal and quadruplex real-time RT-PCR assays using TaqMan-MGB probes were developed for simultaneous detection and differentiation of Chinese PRRSV isolates. The newly developed real-time RT-PCR assays exhibited good specificity, sensitivity, repeatability and reproducibility. In addition, the newly developed real-time RT-PCR assays were further validated by comparing with a universal PRRSV conventional RT-PCR assay on the detection of 664 clinical samples collected from 2016 to 2019 in China. Based on the clinical performance, the agreements between the universal and quadruplex real-time RT-PCR assays and the conventional RT-PCR assay were 99.55% and 99.40%, respectively. Totally 90 samples were detected as PRRSV-positive, including 2 samples that were determined to be co-infected with NL-PRRSV2 and HP-PRRSV2 isolates by the quadruplex real-time RT-PCR assay. ORF5 sequencing confirmed the real-time RT-PCR results that 2, 6, 27 and 57 of the 92 sequences were PRRSV1, CA-PRRSV2, NL-PRRSV2 and HP-PRRSV2, respectively. This study provides promising alternative tools for simultaneous detection and differentiation of PRRSV circulating in Chinese swine herds.

Keywords: ORF5 sequencing; porcine reproductive and respiratory syndrome virus; quadruplex real-time RT-PCR; simultaneous detection and differentiation; universal real-time RT-PCR.

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References

REFERENCES

    1. Bai, X., Wang, Y., Xu, X., Sun, Z., Xiao, Y., Ji, G., … Tian, K. (2016). Commercial vaccines provide limited protection to NADC30-like PRRSV infection. Vaccine, 34, 5540-5545. https://doi.org/10.1016/j.vaccine.2016.09.048
    1. Bustin, S. A., Benes, V., Garson, J. A., Hellemans, J., Huggett, J., Kubista, M., … Wittwer, C. T. (2009). The MIQE guidelines: Minimum information for publication of quantitative real-time PCR experiments. Clinical Chemistry, 55, 611-622. https://doi.org/10.1373/clinchem.2008.112797
    1. Chen, N., Cao, Z., Yu, X., Deng, X., Zhao, T., Wang, L., … Tian, K. (2011). Emergence of novel European genotype porcine reproductive and respiratory syndrome virus in mainland China. The Journal of General Virology, 92, 880-892. https://doi.org/10.1099/vir.0.027995-0
    1. Chen, N. H., Chen, X. Z., Hu, D. M., Yu, X. L., Wang, L. L., Han, W., … Tian, K. G. (2009). Rapid differential detection of classical and highly pathogenic North American Porcine Reproductive and Respiratory Syndrome virus in China by a duplex real-time RT-PCR. Journal of Virological Methods, 161, 192-198. https://doi.org/10.1016/j.jviromet.2009.06.007
    1. Chen, N., Huang, Y., Ye, M., Li, S., Xiao, Y., Cui, B., & Zhu, J. (2019). Co-infection status of classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circoviruses (PCV2 and PCV3) in eight regions of China from 2016 to 2018. Infection, Genetics and Evolution, 68, 127-135. https://doi.org/10.1016/j.meegid.2018.12.011

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