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Volume 25 Issue 1
February 2010
    Citation: Lei CHEN, Xue-zheng FAN, Qin WANG, Lu XU, Qi-zu ZHAO, Yuan-chen ZHOU, Jun LIU, Bo TANG, Xing-qi ZOU. A Novel RT-LAMP Assay for Rapid and Simple Detection of Classical Swine Fever Virus .VIROLOGICA SINICA, 2010, 25(1) : 59-64.  http://dx.doi.org/10.1007/s12250-010-3043-2
    shu

    A Novel RT-LAMP Assay for Rapid and Simple Detection of Classical Swine Fever Virus

    cstr: 32224.14.s12250-010-3043-2

    • China Institute of Veterinary Drug Control, Beijing, 100081, China

    • Corresponding author: Qin WANG, wangqin@ivdc.gov.cn
    • Received Date: 30 March 2009
      Accepted Date: 27 October 2009
      Available online: 01 February 2010

      Fund Project: Beijing Natural Science Foundation 5072041The National Science and Technology supporting plan of the Eleventh Five-year 2006BAD06A18The National Science and Technology supporting plan of the Eleventh Five-year 2006BAD06A03

    • A simple and rapid assay for the detection of Classical swine fever virus (CSFV) was established using reverse transcription loop-mediated isothermal amplification (RT-LAMP). This study describes the amplification of the genomic RNA of CSFV under isothermal conditions (63℃) within one hour, using a set of six primers (two outer primers, two inner primers and two loop primers). This RT-LAMP assay showed 100-fold higher sensitivity than the standard RT-PCR method and identified eighteen additional positive cases that were negative when tested by RT-PCR. This RT-LAMP was able to detect all the 13 strains of CSFV but not the BVDV. PRRSV. SIV. PRV-PCV, thus showed a good specificity. Products amplified by RT-LAMP can be visualized by agarose gel electrophoresis and in addition, either as a white precipitate at the bottom of the tube after a pulse spin or as a color change when dyed with SYBR Green I which are visible to the naked eye. Because RT-LAMP is low-cost and produces rapid results, it has the potential to be an excellent tool for CSFV surveillance in the field, especially in developing countries.
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      1. Anonymous. Council Directive 82/894/EEC of 21 December 1982 on the notification of animal diseases within the Community. Official Journal L, 378: 58-62.

      2. Dukes J P, King D P, Alexandersen S. 2006.Novel reverse transcription loopmediated isothermal amplification for rapid detection of foot-and-mouth disease virus. Arch. Virol, 151: 1093-1106.
          doi: 10.1007/s00705-005-0708-5

      3. Fukuda S, Takao S, Kuwayama M, et al. 2006.Rapid detection of norovirus from fecal specimens by real-time reverse transcription-loop-mediated isothermal amplification assay. J Clin Microbiol. 44: 1376-1381.
          doi: 10.1128/JCM.44.4.1376-1381.2006

      4. Greiser-Wilke I, Blome S, Moenning V. 2007. Diagnostic methods for detection of Classical swine fever virus: Status quo and new developments. Vaccine, 25: 5524-5530.
          doi: 10.1016/j.vaccine.2006.11.043

      5. Marie F L P, Alain M, Philippe V. 2006. Classical swine fever and other Pestiviruses. In: Diseases of Swine, the 9th edition (Straw B E, Zimmerman J, D'Allaire S, et al. ). Blackwell Publishing, p316-317.

      6. Mori Y, Nagamine K, Tomita N, et al. 2001. Detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation. Biochem Biophys Res Commun, 289: 150-154.
          doi: 10.1006/bbrc.2001.5921

      7. Nagamine K, Hase T, Notomi T. 2002. Accelerated reaction by loop-mediated isothermal amplification using loop primers. Mol Cell Probes, 16: 223-229.
          doi: 10.1006/mcpr.2002.0415

      8. Notomi T, Okayama H, Masubuchi H, et al. 2000. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res, 28, e63.
          doi: 10.1093/nar/28.12.e63

      9. Office International des Epizooties, 2006. Diseases notifiable to the OIE; 2006, http://www.oie.int/Eng/maladies/en_classification.htm.

      10. Parida M M, Posadas G, Inoue S, et al. 2004. Real-time reverse transcription loop-mediated isothermal amplification for rapid detection of West Nile virus. J Clin Microbiol, 42: 257-263.
          doi: 10.1128/JCM.42.1.257-263.2004

      11. Parida M M, Horioke K, Ishida H, et al. 2005. Rapid detection and differentiation of dengue virus serotypes by real-time reverse transcription loop-mediated isothermal amplification assay. J Clin Microbiol, 43: 2895-2903.
          doi: 10.1128/JCM.43.6.2895-2903.2005

      12. Parida M M, Santhosh S R, Dash P K, et al. 2006. Development and evaluation of reverse transcription loop-mediated isothermal amplification assay for rapid and real-time detection of Japanese encephalitis virus. J Clin Microbiol, 44 (11): 4172-4178.
          doi: 10.1128/JCM.01487-06

      13. Savan R, Kono T, Itami T, et al. 2005. Loop-mediated isothermal amplification: an emerging technology for detection of fish and shellfish pathogens. J. Fish. Dis. 28: 573-581.

      14. Toriniwa H, Komiya T. 2006. Rapid detection and quantification of Japanese encephalitis virus by real-time reverse transcription loop-mediated isothermal amplification. Microbiol. Immunol. 50: 379-387.
          doi: 10.1111/mim.2006.50.issue-5

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      A Novel RT-LAMP Assay for Rapid and Simple Detection of Classical Swine Fever Virus

        Corresponding author: Qin WANG, wangqin@ivdc.gov.cn
      • China Institute of Veterinary Drug Control, Beijing, 100081, China
      • Received Date: 30 March 2009
      • Accepted Date: 27 October 2009
      Fund Project:  Beijing Natural Science Foundation 5072041The National Science and Technology supporting plan of the Eleventh Five-year 2006BAD06A18The National Science and Technology supporting plan of the Eleventh Five-year 2006BAD06A03

      Abstract: A simple and rapid assay for the detection of Classical swine fever virus (CSFV) was established using reverse transcription loop-mediated isothermal amplification (RT-LAMP). This study describes the amplification of the genomic RNA of CSFV under isothermal conditions (63℃) within one hour, using a set of six primers (two outer primers, two inner primers and two loop primers). This RT-LAMP assay showed 100-fold higher sensitivity than the standard RT-PCR method and identified eighteen additional positive cases that were negative when tested by RT-PCR. This RT-LAMP was able to detect all the 13 strains of CSFV but not the BVDV. PRRSV. SIV. PRV-PCV, thus showed a good specificity. Products amplified by RT-LAMP can be visualized by agarose gel electrophoresis and in addition, either as a white precipitate at the bottom of the tube after a pulse spin or as a color change when dyed with SYBR Green I which are visible to the naked eye. Because RT-LAMP is low-cost and produces rapid results, it has the potential to be an excellent tool for CSFV surveillance in the field, especially in developing countries.

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