DEVELOPMENT AND PRELIMINARY ANALYTICAL EVALUATION OF REAL-TIME PCR ASSAYS FOR AFRICAN SWINE FEVER VIRUS DETECTION
Main Article Content
Authors
R. Nissanova
«Kazakh Scientific Research Veterinary Institute» LLP, Almaty, 223 Rayymbek Avenue, Almaty, 050016, Republic of Kazakhstan
E. Bashenova
«Kazakh Scientific Research Veterinary Institute» LLP, Almaty, 223 Rayymbek Avenue, Almaty, 050016, Republic of Kazakhstan
S. Kaimoldina, Kazakh Scientific Research Veterinary Institute
«Kazakh Scientific Research Veterinary Institute» LLP, Almaty, 223 Rayymbek Avenue, Almaty, 050016, Republic of Kazakhstan
V. Strochkov
«Kazakh Scientific Research Veterinary Institute» LLP, Almaty, 223 Rayymbek Avenue, Almaty, 050016, Republic of Kazakhstan
A. Karabassova
«Kazakh Scientific Research Veterinary Institute» LLP, Almaty, 223 Rayymbek Avenue, Almaty, 050016, Republic of Kazakhstan
P. Akshalova
«Kazakh Scientific Research Veterinary Institute» LLP, Almaty, 223 Rayymbek Avenue, Almaty, 050016, Republic of Kazakhstan
Zh. Abay
«Kazakh Scientific Research Veterinary Institute» LLP, Almaty, 223 Rayymbek Avenue, Almaty, 050016, Republic of Kazakhstan
Zh. Makay
«Kazakh Scientific Research Veterinary Institute» LLP, Almaty, 223 Rayymbek Avenue, Almaty, 050016, Republic of Kazakhstan
Zh. Tlegenova
«Kazakh Scientific Research Veterinary Institute» LLP, Almaty, 223 Rayymbek Avenue, Almaty, 050016, Republic of Kazakhstan
A. Nurpeisova
«Kazakh Scientific Research Veterinary Institute» LLP, Almaty, 223 Rayymbek Avenue, Almaty, 050016, Republic of Kazakhstan
M. Kassenov
«Kazakh Scientific Research Veterinary Institute» LLP, Almaty, 223 Rayymbek Avenue, Almaty, 050016, Republic of Kazakhstan
Abstract
African swine fever virus (ASFV) is a large, double-stranded DNA virus that poses a major threat to the pig industry, highlighting the need for reliable molecular diagnostics. This study reports on the design and analytical development of two primer–probe systems targeting conserved ASFV genomic regions (B646L/p72), supported by in silico assessment and laboratory testing with synthetic DNA constructs. Classical PCR confirmed amplification of both targets from cloned control templates, producing amplicons of the expected size. In probe-based real-time PCR, consistent amplification was obtained for the ASFV-3 system using a pET-asf-3 clone across serial dilutions (Ct ~27–34), while the ASFV-1 probe-based assay did not generate a detectable signal under the applied conditions. Under the tested conditions, the preliminary limit of detection observed for the ASFV-3 probe-based assay was 10³ copies per reaction. These results demonstrate the feasibility of the developed approach and indicate that the ASFV-3 system supports further analytical evaluation, while the ASFV-1 system requires additional optimization of probe parameters and/or thermal cycling conditions.
Keywords
African swine fever virus, real-time PCR, molecular diagnostics, primer–probe design, analytical validation, synthetic DNA controls
Article Details
References
Dixon L.K., Sun H., Roberts H. African swine fever // Antiviral Res. -2019. –Vol. 165. –P. 34-41. - 0.1016/j.antiviral.2019.02.018.
Sánchez-Vizcaíno J.M., Mur L., Martínez-López B. African swine fever: an epidemiological update // Transbound Emerg Dis. – 2012. –Vol. 59. –P. 27-35 - 10.1111/j.1865-1682.2011.01293.x.
Zakharova O.I., Titov I.A., Gogin A.E., Sevskikh T.A., Korennoy F.I., Kolbasov D.V., Abrahamyan L., Blokhin A.A. African Swine Fever in the Russian Far East (2019-2020): Spatio-Temporal Analysis and Implications for Wild Ungulates // Front Vet Sci. - 2021. –Vol. 8. –P. 8:723081 - 10.3389/fvets.2021.723081.
Rock D.L. Challenges for African swine fever vaccine development-"… perhaps the end of the beginning." // Vet Microbiol. – 2017. –Vol. 206. –P. 52-58 - 10.1016/j.vetmic.2016.10.003.
Cappai S., Loi F., Coccollone A., Cocco M., Falconi C., Dettori G., Feliziani F., Sanna M.L., Oggiano A. & Rolesu S. Evaluation of a Commercial Field Test to Detect African Swine Fe-ver // Journal of Wildlife Diseases. - 2017. –Vol. 53 (3). –P. 602–606 - 10.7589/2016-05-112.
Wang L., Luo Y., Zhao Y., Gao G. F., Bi Y., & Qiu H.-J. Comparative genomic analysis reveals an ‘open’ pan-genome of African swine fever virus // Transboundary and Emerging Diseases. - 2020. –Vol. 67(4). –P. 1553–1562 - 10.1111/tbed.13489
Shotin A.R., Mazloum A., Igolkin A.S., Shevchenko I.V., Elsukova A.A., Aronova E.V., Vlasova N.N. Alternative approaches to the diagnosis of African swine fever in the Russian Federa-tion in 2017–2021 // Problems of Virology. - 2022. - Vol. 67. - P. 290-303 - 10.36233/0507-4088-112
Gallardo C., Soler A., Wozniakowski G., & Perez C. A multi gene-approach genotyping method identifies 24 genetic clusters within the genotype II–European African swine fever viruses circulating from 2007 to 2022 // Frontiers in Veterinary Science. - 2023. - Vol. 10. – P. 112233 - 10.3389/fvets.2023.1112850
Wang L., Zhang X., Zhou Y., & Li F. Specific Detection of African Swine Fever Virus Variants: Novel Quadplex Real-Time PCR Assay with Internal Control // Microorganisms. - 2025. - Vol. 13 (3). –P. 615- 622 - 10.3390/microorganisms13030615
Mazur-Panasiuk N., Woźniakowski G. The unique genetic variation within the O174L gene of Polish strains of African swine fever virus facilitates tracking virus origin // Arch Virol. – 2019. - Vol. 164(6). – P. 1667-1672 - 10.1007/s00705-019-04224-x
Quembo C.J., Jori F., Vosloo W., Heath L. Genetic characterization of African swine fe-ver virus isolates from soft ticks at the wildlife/domestic interface in Mozambique and identification of a novel genotype // Transbound Emerg Dis. – 2018. - Vol. 65(2). – P. 420-431 - 10.1111/tbed.12700.
Fernández-Pinero J., Gallardo C., Elizalde M., Robles A., Gómez C., Bishop R., Heath L., Couacy-Hymann E., Fasina F.O., Pelayo V., Soler A., Arias M. Molecular diagnosis of African Swine Fever by a new real-time PCR using universal probe library // Transbound Emerg Dis. – 2013. – Vol. 60(1). – P. 48-58 - 10.1111/j.1865-1682.2012.01317.x.
Bastos A.D., Penrith M.L., Crucière C., Edrich J.L., Hutchings G., Roger F., Couacy-Hymann E. R., Thomson G. Genotyping field strains of African swine fever virus by partial p72 gene characterization // Arch Virol. – 2003. – Vol. 148(4). – P. 693-706 - 10.1007/s00705-002-0946-8.
Agüero M., Fernández J., Romero L., Sánchez Mascaraque C., Arias M., Sánchez-Vizcaíno J.M. Highly sensitive PCR assay for routine diagnosis of African swine fever virus in clini-cal samples // J Clin Microbiol. – 2003. – Vol. 41(9). – P. 4431- 4439 – 10.1128/JCM.41.9.4431-4434.2003.
Tignon M., Gallardo C., Iscaro C., Hutet E., Van der Stede Y., Kolbasov D., De Mia G. M., Le Potier M.F., & Bishop R. P. Development and inter-laboratory validation study of an improved new real-time PCR assay with internal control for detection and laboratory diagnosis of African swine fever virus // Journal of Virological Methods. – Vol. 178 (1–2). –P. 161–170 - 10.1016/j.jviromet.2011.09.007
Alkhamis M.A., Gallardo C., Jurado C., Soler A., Arias M., Sánchez-Vizcaíno J.M. Phy-lodynamics and evolutionary epidemiology of African swine fever p72-CVR genes in Eurasia and Africa // PLoS One. - 2018. – Vol. 13(2). –P. e0192565 - 10.1371/journal.pone.0192565.
Wang L., Li Y., Zhang X., Madera R., Pantua H., Craig A., Muro N., Li D., Retallick J., Ferreyra F.M. Specific Detection of African Swine Fever Virus Variants: Novel Quadplex Real-Time PCR Assay with Internal Control // Microorganisms. – 2025. – Vol. 13(3). –P. 615- 623 - 10.3390/microorganisms13030615
Huang Z., Cao H., Zeng F., Lin S., Chen J., Luo Y., & Zhang G. African swine fever virus MGF505-7R interacts with interferon regulatory factor 9 to evade the type I interferon signaling pathway and promote viral replication // Journal of Virology. – Vol. 97. –P. 1521-1533 - Crossref
Haines F.J., Hofmann M.A., King D.P., Drew T.W., Crooke H.R. Development and Vali-dation of a Multiplex, Real-Time RT PCR Assay for the Simultaneous Detection of Classical and African Swine Fever Viruses // PLoS ONE. – Vol. 8(7): e71019 - Crossref.
OIE (WOAH). African swine fever (infection with African swine fever virus). Terrestrial Manual. Chapter 3.9.1. . - 2022.