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Detection, discrimination and quantitation of 22 bluetongue virus serotypes using real-time RT-PCR with TaqMan MGB probes

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Abstract

Bluetongue virus (BTV) is the etiological agent of bluetongue (BT) disease, a noncontagious insect-transmitted disease of international importance. To date, 26 BTV serotypes have been recognized worldwide. Methods to discriminate BTV serotypes in clinical samples are essential to epidemiological surveillance efforts and BTV vaccination programs. The BTV VP2 major outer capsid protein, encoded by genomic segment 2 (Seg-2), is the most highly variable BTV protein and is the primary determinant of the virus serotype. Here, we report the development of rapid and reliable real-time RT-PCR assays to detect and discriminate 22 BTV serotypes on the basis of VP2-encoding genomic sequences. Serotype-specific primers and probes detected only the targeted BTV serotype and displayed no cross-amplification of off-target BTV serotypes or other closely related Reoviridae and Bunyaviridae family members. The real-time RT-PCR assays developed were highly sensitive, and the majority of serotype-specific reactions could detect template when present at ≥10 copies. These BTV serotype-specific real-time RT-PCR assays represent a rapid, sensitive, and reliable method for the identification, differentiation and quantification of 22 BTV serotypes.

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Acknowledgments

The authors thank Dr. Peter Wilker for editing the manuscript. This study was supported by grants from the National Natural Science Foundation of China (No. 31402207), Basic Scientific Research Foundation of Central Research Academies and Institutes (ZGKJ1610302015008) and National High-Tech Research and Development Program of China (No. 2011AA10A212).

Conflict of interest

None of the authors has any financial or personal relationships that could inappropriately influence or bias the content of the paper.

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Authors and Affiliations

  1. Department of Basic Veterinary Medicine, College of Veterinary Medicine, Northeast Agricultural University, 59 Mucai Street, Xiangfang District, Harbin, 150030, People’s Republic of China

    Yufei Feng & Donglai Wu

  2. The Key Laboratory of Veterinary Public Health, Ministry of Agriculture, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, 150001, People’s Republic of China

    Yufei Feng, Tao Yang, Qingyuan Xu, Encheng Sun, Junping Li, Shuang Lv, Haixiu Wang, Qin Zhang, Jikai Zhang & Donglai Wu

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  1. Yufei Feng
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  2. Tao Yang
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Correspondence to Donglai Wu.

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Feng, Y., Yang, T., Xu, Q. et al. Detection, discrimination and quantitation of 22 bluetongue virus serotypes using real-time RT-PCR with TaqMan MGB probes. Arch Virol 160, 2249–2258 (2015). https://doi.org/10.1007/s00705-015-2499-7

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  • DOI: https://doi.org/10.1007/s00705-015-2499-7

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