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Citation: Tianli Zou, Junhua Deng, Xiangdong Li, Shiyin Zhang, Lingyan Chen, Liying Hao, Jinshan Zhuang, Heng Wang, Guihong Zhang, Shengxiang Ge, Kegong Tian. Development of a fluorescent probe hydrolysis-insulated isothermal PCR for rapid and sensitive on-site detection of African swine fever virus [J].VIROLOGICA SINICA, 2022, 37(3) : 462-464.  http://dx.doi.org/10.1016/j.virs.2022.03.002

Development of a fluorescent probe hydrolysis-insulated isothermal PCR for rapid and sensitive on-site detection of African swine fever virus

  • Corresponding author: Shengxiang Ge, sxge@xmu.edu.cn
    Kegong Tian, tiankg@263.net
  • Received Date: 11 November 2021
    Accepted Date: 01 March 2022
    Available online: 04 March 2022
  • Highlights
    1. A probe-based insulated isothermal PCR (iiPCR) assay was developed for rapid and onsite detection of ASFV.
    2. The developed iiPCR showed similar sensitivity and specificity with OIE recommended real-time PCR.
    3. Blood samples could be directly applied as PCR template in iiPCR without DNA extraction.

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  • 10.1016j.virs.2022.03.002-ESM.docx
    1. Ambagala, A., Fisher, M., Goolia, M., Nfon, C., Furukawa-Stoffer, T., Ortega Polo, R., Lung, O., 2017. Field-deployable reverse transcription-insulated isothermal PCR (RTiiPCR) assay for rapid and sensitive detection of foot-and-mouth disease virus. Transboundary Emerg. Dis. 64, 1610-1623.

    2. Chen, N., Ye, M., Xiao, Y., Li, S., Huang, Y., Li, X., Tian, K., Zhu, J., 2019. Development of universal and quadruplex real-time RT-PCR assays for simultaneous detection and differentiation of porcine reproductive and respiratory syndrome viruses. Transboundary Emerg. Dis. 66, 2271-2278.

    3. Gaudreault, N.N., Richt, J.A., 2019. Subunit vaccine approaches for African swine fever virus. Vaccines 7, 56.

    4. James, H.E., Ebert, K., McGonigle, R., Reid, S.M., Boonham, N., Tomlinson, J.A., Hutchings, G.H., Denyer, M., Oura, C.A., Dukes, J.P., King, D.P., 2010. Detection of African swine fever virus by loop-mediated isothermal amplification. J. Virol Methods 164, 68-74.

    5. Li, X., Tian, K., 2018. African swine fever in China. Vet. Rec. 183, 300-301.

    6. Lung, O., Pasick, J., Fisher, M., Buchanan, C., Erickson, A., Ambagala, A., 2016. Insulated isothermal reverse transcriptase PCR (iiRT-PCR) for rapid and sensitive detection of classical swine fever virus. Transboundary Emerg. Dis. 63, e395-402.

    7. Luo, Y., Atim, S.A., Shao, L., Ayebazibwe, C., Sun, Y., Liu, Y., Ji, S., Meng, X.Y., Li, S., Li, Y., Masembe, C., Stahl, K., Widen, F., Liu, L., Qiu, H.J., 2017. Development of an updated PCR assay for detection of African swine fever virus. Arch. Virol. 162, 191-199.

    8. Miao, F., Zhang, J., Li, N., Chen, T., Wang, L., Zhang, F., Mi, L., Wang, S., Wang, Y., Zhou, X., Zhang, Y., Li, M., Zhang, S., Hu, R., 2019. Rapid and sensitive recombinase polymerase amplification combined with lateral flow strip for detecting African swine fever virus. Front. Microbiol. 10, 1004.

    9. Sanchez, E.G., Perez-Nunez, D., Revilla, Y., 2019. Development of vaccines against African swine fever virus. Virus Res. 265, 150-155.

    10. Tran, H.N.T., Le, H.C.T., Pham, B.P., Luu, V.Q., Nguye, V.L., 2021. Evaluation of an automated insulated isothermal polymerase chain reaction system for rapid and reliable, on-site detection of African swine fever virus. J. Am. Vet. Med. Assoc. 15, 662-668.

    11. Tsai, Y.L., Wang, H.T., Chang, H.F., Tsai, C.F., Lin, C.K., Teng, P.H., Su, C., Jeng, C.C., Lee, P.Y., 2012. Development of TaqMan probe-based insulated isothermal PCR (iiPCR) for sensitive and specific on-site pathogen detection. PLoS One 7, e45278.

    12. Zhang, J., Nfon, C., Tsai, C.F., Lee, C.H., Fredericks, L., Chen, Q., Sinha, A., Bade, S., Harmon, K., Pineyro, P., Gauger, P., Tsai, Y.L., Wang, H.T., Lee, P.A., 2019. Development and evaluation of a real-time RT-PCR and a field-deployable RTinsulated isothermal PCR for the detection of Seneca Valley virus. BMC Vet. Res. 15, 168.

    13. Zhao, D., Liu, R., Zhang, X., Li, F., Wang, J., Zhang, J., Liu, X., Wang, L., Wu, X., Guan, Y., Chen, W., Wang, X., He, X., Bu, Z., 2019. Replication and virulence in pigs of the first African swine fever virus isolated in China. Emerg. Microb. Infect. 8, 438-447.

    14. Zhou, X., Li, N., Luo, Y., Liu, Y., Miao, F., Chen, T., Zhang, S., Cao, P., Li, X., Tian, K., Qiu, H.J., Hu, R., 2018. Emergence of African swine fever in China, 2018. Transboundary Emerg. Dis. 65, 1482-1484.

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    Development of a fluorescent probe hydrolysis-insulated isothermal PCR for rapid and sensitive on-site detection of African swine fever virus

      Corresponding author: Shengxiang Ge, sxge@xmu.edu.cn
      Corresponding author: Kegong Tian, tiankg@263.net
    • a Beijing Wantai Biopharmaceutical Co., Ltd, Beijing, 102206, China;
    • b Luoyang Putai Bio-Tech Co. Ltd, Luoyang, 471003, China;
    • c National Research Center for Veterinary Medicine, Luoyang, 471003, China;
    • d School of Public Health, Xiamen University, Xiamen, 361102, China;
    • e ASFV Regional Reference Laboratory in Guangzhou, South China Agricultural University, Guangzhou, 510642, China

    Abstract: Highlights
    1. A probe-based insulated isothermal PCR (iiPCR) assay was developed for rapid and onsite detection of ASFV.
    2. The developed iiPCR showed similar sensitivity and specificity with OIE recommended real-time PCR.
    3. Blood samples could be directly applied as PCR template in iiPCR without DNA extraction.

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