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. doi: 10.1016/j.virs.2022.03.002
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

利用热对流PCR技术快速灵敏检测非洲猪瘟病毒的研究

  • 在过去几年里,非洲猪瘟一直困扰着亚洲生猪产业,给养猪业造成了巨大的经济损失。快速、灵敏的核酸检测方法是实施严格的扑杀政策以控制该病的前提条件。在这项研究中,我们开发了一种新型荧光热对流PCR(iiPCR)检测方法,用于快速和现场ASFV的检测。该方法显示出良好的灵敏度和特异性。经国家非洲猪瘟区域参考实验室验证56份DNA样品,本研究中建立的iiPCR方法与OIE推荐的real-time PCR方法符合率为100%。此外,该方法检测血液样品无需核酸提取,可直接作为模板进行现场快速检测。

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
  • 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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    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.

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    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.

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    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.

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    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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  • 10.1016j.virs.2022.03.002-ESM.docx

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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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