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Development of a fluorescent-intercalating-dye-based reverse transcription loop-mediated isothermal amplification assay for rapid detection of seasonal Japanese B encephalitis outbreaks in pigs

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Abstract

The standardization and validation of a one-step, single-tube, accelerated fluorescent-intercalating-dye-based reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay targeting the NS3 gene of Japanese B encephalitis virus (JEV) is described for rapid, simple, and high-throughput detection of JEV. The amplification can be completed in 35 min under isothermal conditions at 63°C by employing a set of six primers targeting the NS3 gene of JEV. The RT-LAMP assay described demonstrated high sensitivity for detecting JEV, with a detection limit in swine samples of 8.13 PFU/ml. The specificity of the selected primer sets was established by cross-reactivity studies with pathogens that exhibit similar clinical signs and testing of samples from healthy animals. The clinical applicability of the RT-LAMP assay was validated using either spiked samples or samples from seasonal outbreaks. The comparative evaluation of the RT-LAMP assay revealed 79.59 % concordance with conventional RT-PCR targeting the E gene of JEV. The RT-LAMP assay reported here is a valuable tool for rapid real-time and high-throughput seasonal infection surveillance and quarantine after outbreak through blood sampling by using ordinary real-time PCR thermocyclers without purchasing an expensive Loopamp real-time turbidimeter.

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Acknowledgments

This work was supported in part by grants from the AQSIQ Project of China (No. 2008IK262, and 2009GDK16). This study was also supported by AQSIQ Quality Public Project of China (No. 10-65).

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Authors and Affiliations

  1. Guangdong Inspection and Quarantine Technology Center, Guangzhou, Guangdong, 510623, China

    C. J. Tian, Z. X. Lin, Q. Luo, C. B. Luo, H. Q. Yu, R. Chen, X. W. Wu & D. Z. Zhu

  2. South China Agricultural University, Guangzhou, Guangdong, 510642, China

    X. M. He, Y. Z. Bi & J. Ji

  3. Department of Biotech, Expression BioSciences. LLC, Newark, DE, 19713, USA

    Z. J. Ren

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  1. C. J. Tian
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  2. Z. X. Lin
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  3. X. M. He
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  4. Q. Luo
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  5. C. B. Luo
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  12. J. Ji
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Correspondence to C. J. Tian.

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Tian, C.J., Lin, Z.X., He, X.M. et al. Development of a fluorescent-intercalating-dye-based reverse transcription loop-mediated isothermal amplification assay for rapid detection of seasonal Japanese B encephalitis outbreaks in pigs. Arch Virol 157, 1481–1488 (2012). https://doi.org/10.1007/s00705-012-1330-y

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  • DOI: https://doi.org/10.1007/s00705-012-1330-y

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