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A loop-mediated isothermal amplification method for the detection of members of the genus Ranavirus

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Abstract

A loop-mediated isothermal amplification (LAMP) method was developed for detection of members of the genus Ranavirus. The optimum reaction mixture contained 2.5 μL of each inner primer, RV-FIP (20 pmol/μL) and RV-BIP (20 pmol/μL), 0.5 μL of each outer primer, RV-F3 (10 pmol/μL) and RV-B3 (10 pmol/μL), 1.25 μL of each loop primer, RV-LF (20 pmol/μL) and RV-LB (20 pmol/μL), 3.5 μL dNTP mix (10 mM each), 8 μL MgSO4 (25 mM), 1 μL of Bst DNA polymerase (8 U/mL, large fragment; New England Biolabs Inc., Beverly, MA, USA), 2.5 μL 10 × supplied buffer, and 1 μL of template DNA in a final volume of 25 μL. The optimum reaction conditions were 63 °C for 60 min. This LAMP method could detect Andrias davidianus iridovirus (ADIV), soft-shelled turtle iridovirus (STIV), and epizootic hematopoietic necrosis virus (EHNV), all of which belong to the genus Ranavirus, but it could not detect other viruses such as koi herpes virus (KHV), channel catfish virus (CCV), infectious spleen and kidney necrosis virus (ISKNV) and white spot syndrome virus (WSSV). The detection limit of the LAMP method was 100 copies of STIV DNA segment, and the sensitivity was 10 times higher than that of the polymerase chain reaction (PCR) assay. The results could be estimated visually by eye when calcein was added.

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Acknowledgments

This study was supported by the Public Welfare Project of General Administration of Quality Supervision, Inspection and Quarantine of P. R. C (AQSIQ) (201010020), and the Fundamental Research Funds for the Public Research Institutes (2010JK003).

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Authors and Affiliations

  1. The Institute of Animal Quarantine, Chinese Academy of Inspection and Quarantine, No.241 Building, Huixinxijie, Chaoyang District, Beijing, 100029, China

    Zhang Min, Jing Hongli, Wang Na, Wu Shaoqiang & Lin Xiangmei

  2. Beijing Exit-Entry Inspection and Quarantine Bureau, No.6 Building, Tianshuiyuanjie, Chaoyang District, Beijing, 100026, China

    Zhang Lifeng

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  1. Zhang Min
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  2. Jing Hongli
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  3. Zhang Lifeng
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Correspondence to Zhang Min or Lin Xiangmei.

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Min, Z., Hongli, J., Lifeng, Z. et al. A loop-mediated isothermal amplification method for the detection of members of the genus Ranavirus . Arch Virol 158, 2121–2126 (2013). https://doi.org/10.1007/s00705-013-1724-5

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  • DOI: https://doi.org/10.1007/s00705-013-1724-5

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