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Pathogens
Volume 14
Issue 5
10.3390/pathogens14050473
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This is an early access version, the complete PDF, HTML, and XML versions will be available soon.
Article

Simultaneous Detection of Classical and African Swine Fever Viruses by Duplex Taqman Real-Time PCR Assay in Pigs Infected with Both Diseases

by
Liani Coronado
1,2,3,
Adriana Muñoz-Aguilera
1,2,3,4,
Miaomiao Wang
1,2,3,
Iván Muñoz
1,2,3,
Cristina Riquelme
1,2,3,
Saray Heredia
1,2,3,
Katarzyna Stępniewska
5,
Carmina Gallardo
6 and
Llilianne Ganges
1,2,3,*
1
WOAH Reference Laboratory for Classical Swine Fever, IRTA-CReSA, 08193 Barcelona, Spain
2
Unitat Mixta d’Investigació IRTA-UAB en Sanitat Animal, Centre de Recerca en Sanitat Animal (CReSA), Bellaterra, 08193 Barcelona, Spain
3
IRTA, Programa de Sanitat Animal, Centre de Recerca en Sanitat Animal (CReSA), Bellaterra, 08193 Barcelona, Spain
4
Instituto Colombiano Agropecuario (ICA), Bogotá 110911, Colombia
5
Department of Swine Diseases, National Veterinary Research Institute, Partyzantów 57 Avenue, 24-100 Pulawy, Poland
6
European Union Reference Laboratory for African Swine Fever (EURL), Centro de Investigación en Sanidad Animal (CISA), Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Consejo Superior de Investigaciones Científicas (CSIC), Valdeolmos, 28130 Madrid, Spain
*
Author to whom correspondence should be addressed.
Pathogens 2025, 14(5), 473; https://doi.org/10.3390/pathogens14050473
Submission received: 8 April 2025 / Revised: 6 May 2025 / Accepted: 9 May 2025 / Published: 13 May 2025
(This article belongs to the Section Viral Pathogens)
Versions Notes

Abstract

The increasing spread of African swine fever (ASF) in recent years and the presence of classical swine fever (CSF) subclinical forms in endemic countries suggests that the possibility of coinfection with ASF virus (ASFV) and CSF virus (CSFV) in pigs cannot be ruled out in areas where both diseases are prevalent. Thus, rapid and reliable diagnosis through molecular testing is essential for the timely implementation of control measures to prevent the spread of these devastating swine diseases. Here, we have coupled two of the most validated PCR assays for the detection of CSFV and ASFV in a single reaction tube. The combination of the two tests for the detection of two target nucleic acids did not affect the analytical sensitivity, and the duplex RT-qPCR assay was comparable with the standard molecular techniques. The detection limits for CSFV RNA and ASFV DNA were 0.12 TCID50/reaction and 0.25 TCID50/reaction, respectively. The test showed high repeatability and reproducibility, the coefficient of variation was below 2%, and excellent performance was demonstrated in clinical samples. The duplex assay shows great potential to become a robust diagnostic tool for the rapid and reliable detection and differentiation of CSFV and ASFV in areas where both viruses may be circulating.
Keywords: early diagnosis; differential detection; duplex qPCR; CSFV; ASFV; doubly infected pigs; surveillance early diagnosis; differential detection; duplex qPCR; CSFV; ASFV; doubly infected pigs; surveillance

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MDPI and ACS Style

Coronado, L.; Muñoz-Aguilera, A.; Wang, M.; Muñoz, I.; Riquelme, C.; Heredia, S.; Stępniewska, K.; Gallardo, C.; Ganges, L. Simultaneous Detection of Classical and African Swine Fever Viruses by Duplex Taqman Real-Time PCR Assay in Pigs Infected with Both Diseases. Pathogens 2025, 14, 473. https://doi.org/10.3390/pathogens14050473

AMA Style

Coronado L, Muñoz-Aguilera A, Wang M, Muñoz I, Riquelme C, Heredia S, Stępniewska K, Gallardo C, Ganges L. Simultaneous Detection of Classical and African Swine Fever Viruses by Duplex Taqman Real-Time PCR Assay in Pigs Infected with Both Diseases. Pathogens. 2025; 14(5):473. https://doi.org/10.3390/pathogens14050473

Chicago/Turabian Style

Coronado, Liani, Adriana Muñoz-Aguilera, Miaomiao Wang, Iván Muñoz, Cristina Riquelme, Saray Heredia, Katarzyna Stępniewska, Carmina Gallardo, and Llilianne Ganges. 2025. "Simultaneous Detection of Classical and African Swine Fever Viruses by Duplex Taqman Real-Time PCR Assay in Pigs Infected with Both Diseases" Pathogens 14, no. 5: 473. https://doi.org/10.3390/pathogens14050473

APA Style

Coronado, L., Muñoz-Aguilera, A., Wang, M., Muñoz, I., Riquelme, C., Heredia, S., Stępniewska, K., Gallardo, C., & Ganges, L. (2025). Simultaneous Detection of Classical and African Swine Fever Viruses by Duplex Taqman Real-Time PCR Assay in Pigs Infected with Both Diseases. Pathogens, 14(5), 473. https://doi.org/10.3390/pathogens14050473

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