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Volume 26 Issue 4
August 2011
    Citation: Lei Zhang, Ye-bing Liu, Lei Chen, Jian-huan Wang, Yi-bao Ning. Rapid and Sensitive Detection of PRRSV by a Reverse Transcription-Loop-mediated Isothermal Amplification Assay .VIROLOGICA SINICA, 2011, 26(4) : 252-259.  http://dx.doi.org/10.1007/s12250-011-3185-x
    shu

    Rapid and Sensitive Detection of PRRSV by a Reverse Transcription-Loop-mediated Isothermal Amplification Assay

    cstr: 32224.14.s12250-011-3185-x
    • 1.

      China Institute of Veterinary Drug Control, Beijing 100081, China

    • 2.

      College of Veterinary Medicine, Northwest A & F University, Yangling, Shaanxi 712100, China

    • Corresponding author: Yi-bao Ning, ningyibao@sina.com
    • These authors contributed equally
    • Received Date: 24 January 2011
      Accepted Date: 11 May 2011
      Available online: 01 August 2011
    • A real-time monitoring reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the sensitive and specific detection of prototypic, prevalent North American porcine reproductive and respiratory syndrome virus (PRRSV) strains. As a higher sensitivity and specificity method than reverse transcription polymerase chain reaction (RT-PCR), the RT-LAMP method only used a turbidimeter, exhibited a detection limit corresponding to a 10–4 dilution of template RNA extracted from 250 μL of 105 of the 50% tissue culture infective dose (TCID50) of PRRSV-containing cells, and no cross-reactivity was observed with other related viruses including porcine circovirus type 2, swine influenza virus, porcine rotavirus and classical swine fever virus. From forty-two field samples, 33 samples in the RT-LAMP assay was detected positive, whereas three of which were not detected by RT-PCR. Furthermore, in 33 strains of PRRSV, an identical detection rate was observed with the RT-LAMP assay to what were isolated using porcine alveolar macrophages.These findings demonstrated that the RT-LAMP assay has potential clinical applications for the detection of highly pathogenic PRRSV isolates, especially in developing countries.
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      3. Hong T C, Mai Q L, Cuong D V, et al. 2004. Development and evaluation of a novel loop-mediated isothermal amplification method for rapid detection of severe acute respiratory syndrome coronavirus. J Clin Microbiol, 43: 1956-1961.

      4. Imai M, Ninomiya A, Minekawa H, et al. 2006. Development of HS-RT-LAMP (loop-mediated isothermal amplification) system for rapid diagnosis of H5 avian influenza virus infection. Vaccine, 24(44/46): 6679-6682.

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      7. Larochelle R, Magar R. 1995. Comparison of immunogold silver staining (IGSS) with two immunoperoxidase staining systems for the detection of porcine reproductive and respiratory syndrome virus (PRRSV) antigens in formalin-fixed tissues. J Vet Diagn Invest, 7: 540-543.
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      Rapid and Sensitive Detection of PRRSV by a Reverse Transcription-Loop-mediated Isothermal Amplification Assay

        Corresponding author: Yi-bao Ning, ningyibao@sina.com
      • 1. China Institute of Veterinary Drug Control, Beijing 100081, China
      • 2. College of Veterinary Medicine, Northwest A & F University, Yangling, Shaanxi 712100, China
      • Received Date: 24 January 2011
      • Accepted Date: 11 May 2011

      Abstract: A real-time monitoring reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the sensitive and specific detection of prototypic, prevalent North American porcine reproductive and respiratory syndrome virus (PRRSV) strains. As a higher sensitivity and specificity method than reverse transcription polymerase chain reaction (RT-PCR), the RT-LAMP method only used a turbidimeter, exhibited a detection limit corresponding to a 10–4 dilution of template RNA extracted from 250 μL of 105 of the 50% tissue culture infective dose (TCID50) of PRRSV-containing cells, and no cross-reactivity was observed with other related viruses including porcine circovirus type 2, swine influenza virus, porcine rotavirus and classical swine fever virus. From forty-two field samples, 33 samples in the RT-LAMP assay was detected positive, whereas three of which were not detected by RT-PCR. Furthermore, in 33 strains of PRRSV, an identical detection rate was observed with the RT-LAMP assay to what were isolated using porcine alveolar macrophages.These findings demonstrated that the RT-LAMP assay has potential clinical applications for the detection of highly pathogenic PRRSV isolates, especially in developing countries.

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