Development of a loop-mediated isothermal amplification assay for rapid detection of African swine fever https://doi.org/10.12982/VIS.2021.008

Main Article Content

Arphaphorn Dokphut
Prakit Boonpornprasert
Tapanut Songkasupa
Supansa Tangdee

Abstract

Since the first African swine fever (ASF) outbreak was reported in China in 2018, the disease has spread rapidly to several countries in Asia. The early detection of this disease is essential for the ASF control strategy to be effective. Loop-mediated isothermal amplification (LAMP) is a nucleic acid detection assay that is rapid, simple, cost-effective and field-friendly. In this study, we have developed a colorimetric assay of LAMP to detect ASF virus (ASFV). A set of LAMP primers was designed to target the conserved region of the VP72 gene. The conditions of LAMP were optimized. The amplification products were easily detected by the naked eye using hydroxynaphthol blue (HNB). The positive LAMP reaction generated a violet to sky blue color change. The sensitivity and specificity of LAMP assay were demonstrated in comparison with the OIE-recommended real-time PCR. A total of 211 samples including 121 confiscated pork products and 90 spiked clinical specimens were tested. The optimal amplification of ASFV DNA by LAMP was incubation at 60 °C for 90 min. The analytical sensitivity of ASFV LAMP assay was at least 368 plasmid DNA copies/µL without cross-reactivity with other swine pathogens. The diagnostic sensitivity and specificity of LAMP were 88% and 100%, respectively. There was almost perfect agreement between LAMP and real-time PCR assays (Kappa value=0.84). This novel LAMP assay is deemed to be a rapid, simple, sensitive, specific diagnostic tool and suitable for early detection of ASF to minimize the likelihood of ASF spread nationwide.

Downloads

Download data is not yet available.

Article Details

How to Cite
Dokphut, A., Boonpornprasert, P., Songkasupa, T., & Tangdee, S. (2020). Development of a loop-mediated isothermal amplification assay for rapid detection of African swine fever: https://doi.org/10.12982/VIS.2021.008. Veterinary Integrative Sciences, 19(1), 87–100. Retrieved from https://he02.tci-thaijo.org/index.php/vis/article/view/246878
Section
Research Articles

References

Aguero, M., Fernandez, J., Romero, L., Sanchez Mascaraque, C., Arias, M., Sanchez-Vizcaino, J.M., 2003. Highly sensitive PCR assay for routine diagnosis of African swine fever virus in clinical samples. J Clin Microbiol 41, 4431-4434.
Alonso, C., Borca, M., Dixon, L., Revilla, Y., Rodriguez, F., Escribano, J.M., Ictv Report, C., 2018. ICTV Virus Taxonomy Profile: Asfarviridae. J Gen Virol 99, 613-614.
Arias, M., Jurado, C., Gallardo, C., Fernandez-Pinero, J., Sanchez-Vizcaino, J.M., 2018. Gaps in African swine fever: Analysis and priorities. Transbound Emerg Dis 65 Suppl 1, 235-247.
Atuhaire, D.K., Afayoa, M., Ochwo, S., Katiti, D., 2014. Comparative detection of African swine fever virus by loop-mediated isothermal amplification assay and polymerase chain reaction in domestic pigs in Uganda. African J Microbiol Res 8, 2322-2328.
Choi, E.J., Lee, C.H., Song, J.Y., Song, H.J., Park, C.K., Kim, B., Shin, Y.K., 2013. Genetic diversity of porcine reproductive and respiratory syndrome virus in Korea. J Vet Sci 14, 115-124.
Das, A., Babiuk, S., McIntosh, M.T., 2012. Development of a loop-mediated isothermal amplification assay for rapid detection of capripoxviruses. J Clin Microbiol 50, 1613-1620.
Dehghan Esmatabadi, M.J., Bozorgmehr, A., Motalebzadeh, H., Bodaghabadi, N., Farhangi, B., Babashah, S., Sadeghizadeh, M., 2015. Techniques for Evaluation of LAMP Amplicons and their Applications in Molecular Biology. Asian Pac J Cancer Prev 16, 7409-7414.
Drew, T.W., Lowings, J.P., Yapp, F., 1997. Variation in open reading frames 3, 4 and 7 among porcine reproductive and respiratory syndrome virus isolates in the UK. Vet Microbiol 55, 209-221.
Duan, Y., Zhang, X., Ge, C., Wang, Y., Cao, J., Jia, X., Wang, J., Zhou, M., 2014. Development and application of loop-mediated isothermal amplification for detection of the F167Y mutation of carbendazim-resistant isolates in Fusarium graminearum. Sci Rep 4, 7094.
Ellis, J., Krakowka, S., Lairmore, M., Haines, D., Bratanich, A., Clark, E., Allan, G., Konoby, C., Hassard, L., Meehan, B., Martin, K., Harding, J., Kennedy, S., McNeilly, F., 1999. Reproduction of lesions of postweaning multisystemic wasting syndrome in gnotobiotic piglets. J Vet Diagn Invest 11, 3-14.
Fernandez-Pinero, J., Gallardo, C., Elizalde, M., Robles, A., Gomez, C., Bishop, R., Heath, L., Couacy-Hymann, E., Fasina, F.O., Pelayo, V., Soler, A., Arias, M., 2013. Molecular diagnosis of African Swine Fever by a new real-time PCR using universal probe library. Transbound Emerg Dis 60, 48-58.
Francois, P., Tangomo, M., Hibbs, J., Bonetti, E.J., Boehme, C.C., Notomi, T., Perkins, M.D., Schrenzel, J., 2011. Robustness of a loop-mediated isothermal amplification reaction for diagnostic applications. FEMS Immunol Med Microbiol 62, 41-48.
Gallardo, C., Fernandez-Pinero, J., Arias, M., 2019. African swine fever (ASF) diagnosis, an essential tool in the epidemiological investigation. Virus Res 271, 197676.
Goto, M., Honda, E., Ogura, A., Nomoto, A., Hanaki, K., 2009. Colorimetric detection of loop-mediated isothermal amplification reaction by using hydroxy naphthol blue. Biotechniques 46, 167-172.
Hoffmann, E., Stech, J., Guan, Y., Webster, R.G., Perez, D.R., 2001. Universal primer set for the full-length amplification of all influenza A viruses. Arch Virol 146, 2275-2289.
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.
Khan, M., Li, B., Jiang, Y., Weng, Q., Chen, Q., 2017. Evaluation of Different PCR-Based Assays and LAMP Method for Rapid Detection of Phytophthora infestans by Targeting the Ypt1 Gene. Front Microbiol 8, 1920.
King, D.P., Reid, S.M., Hutchings, G.H., Grierson, S.S., Wilkinson, P.J., Dixon, L.K., Bastos, A.D., Drew, T.W., 2003. Development of a TaqMan PCR assay with internal amplification control for the detection of African swine fever virus. J Virol Methods 107, 53-61.
Kweon, C.H., Lee, J.G., Han, M.G., Kang, Y.B., 1997. Rapid diagnosis of porcine epidemic diarrhea virus infection by polymerase chain reaction. J Vet Med Sci 59, 231-232.
Landis, J.R., Koch, G.G., 1977. The measurement of observer agreement for categorical data. Biometrics 33, 159-174.
Lowings, P., Ibata, G., Needham, J., Paton, D., 1996. Classical swine fever virus diversity and evolution. J Gen Virol 77 ( Pt 6), 1311-1321.
Notomi, T., Okayama, H., Masubuchi, H., Yonekawa, T., Watanabe, K., Amino, N., Hase, T., 2000. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res 28, E63.
Paton, D., Ibata, G., Sands, J., McGoldrick, A., 1997. Detection of transmissible gastroenteritis virus by RT-PCR and differentiation from porcine respiratory coronavirus. J Virol Methods 66, 303-309.
Quembo, C.J., Jori, F., Vosloo, W., Heath, L., 2018. Genetic characterization of African swine fever virus isolates from soft ticks at the wildlife/domestic interface in Mozambique and identification of a novel genotype. Transbound Emerg Dis 65, 420-431.
Reid, S.M., Ferris, N.P., Hutchings, G.H., Samuel, A.R., Knowles, N.J., 2000. Primary diagnosis of foot-and-mouth disease by reverse transcription polymerase chain reaction. J Virol Methods 89, 167-176.
Songkasupa, T., Dokphut, A., Boonpornprasert, P., 2020. Detection of African swine fever virus in confiscated pork products brought into Thailand during 2018-2019. Thai J Vet Med 50 Suppl, 257-259.
Wang, X., Seo, D.J., Lee, M.H., Choi, C., 2014. Comparison of conventional PCR, multiplex PCR, and loop-mediated isothermal amplification assays for rapid detection of Arcobacter species. J Clin Microbiol 52, 557-563.
Whelan, J.A., Russell, N.B., Whelan, M.A., 2003. A method for the absolute quantification of cDNA using real-time PCR. J Immunol Methods 278, 261-269.
World Organisation for Animal Health (OIE), 2019. Global situation of ASF report. https://www.oie.int/fileadmin/Home/eng/Animal_Health_in_the_World/docs/pdf/Disease_cards/ASF/Report_17._Global_situation_of_ASF.pdf. Accessed 20 Dec 2019.
World Organisation for Animal Health (OIE), 2020. African swine fever. In: Manual of Diagnostic Tests and Vaccines for Terrestrial Animals 2019 Chapter 3.8.1. http://www.oie.int/fileadmin/Home/eng/Health_standards/tahm/3.08.01_ASF.pdf. Accessed 27 June 2020.
Wu, X., Xiao, L., Wang, Y., Yang, Z., Yao, X., Peng, B., 2016. Development of a rapid and sensitive method for detection of African swine fever virus using loop-mediated isothermal amplification. Braz. Arch. Biol. Technol. 59, e16160500.
Zhang, S.Y., Dai, D.J., Wang, H.D., Zhang, C.Q., 2019. One-step loop-mediated isothermal amplification (LAMP) for the rapid and sensitive detection of Fusarium fujikuroi in bakanae disease through NRPS31, an important gene in the gibberellic acid bio-synthesis. Sci Rep 9, 3726.
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. Transbound Emerg Dis 65, 1482-1484.