Development of an updated PCR assay for detection of African swine fever virus
Due to the current unavailability of vaccines or treatments for African swine fever (ASF), which is caused by African swine fever virus (ASFV), rapid and reliable detection of the virus is essential for timely implementation of emergency control measures and differentiation of ASF from other swine diseases with similar clinical presentations. Here, an improved PCR assay was developed and evaluated for sensitive and universal detection of ASFV. Primers specific for ASFV were designed based on the highly conserved region of the vp72 gene sequences of all ASFV strains available in GenBank, and the PCR assay was established and compared with two OIE-validated PCR tests. The analytic detection limit of the PCR assay was 60 DNA copies per reaction. No amplification signal was observed for several other porcine viruses. The novel PCR assay was more sensitive than two OIE-validated PCR assays when testing 14 strains of ASFV representing four genotypes (I, V, VIII and IX) from diverse geographical areas. A total of 62 clinical swine blood samples collected from Uganda were examined by the novel PCR, giving a high agreement (59/62) with a superior sensitive universal probe library-based real-time PCR. Eight out of 62 samples tested positive, and three samples with higher Ct values (39.15, 38.39 and 37.41) in the real-time PCR were negative for ASFV in the novel PCR. In contrast, one (with a Ct value of 29.75 by the real-time PCR) and two (with Ct values of 29.75 and 33.12) ASFV-positive samples were not identified by the two OIE-validated PCR assays, respectively. Taken together, these data show that the novel PCR assay is specific, sensitive, and applicable for molecular diagnosis and surveillance of ASF.
KeywordsWild Boar Classical Swine Fever Virus African Swine Fever African Swine Fever Virus Wild Boar Population
We are grateful to Dr. Mo Zhou at Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, for revising the manuscript.
Compliance with ethical standards
This work was supported by grants from the European Union’s Seventh Framework Programme LinkTADs (No. 613804), the Harbin Animal Husbandry and Veterinary Bureau of China, and the OIE twinning project between SVA, Sweden and MAAIF-NADDEC, Uganda. Charles Masembe was funded by the Wellcome Trust Fellowship.
Conflict of interest
All authors declare that they have no competing interests.
None of the experiments in this study involved human participants.
- 3.Denyer MS, Wilkinson PJ (1998) African swine fever. In: Encyclopedia of immunology, pp 54–56Google Scholar
- 6.World Organization for Animal Health (OIE) (2014) World animal health information database (WAHID). http://www.oie.int/wahis_2/public/wahid.php/Diseaseinformation/Immsummary
- 9.Food and Agriculture Organization, United Nations (2013) African swine fever in the Russian Federation: risk factors for Europe and beyond. EMPRES WATCH, vol. 28. http://www.fao.org/docrep/018/aq240e/aq240e.pdf
- 13.Oura CA, Arias M (2012) African swine fever. In: OIE Biological Standards Commission (ed) OIE Manual of diagnostic tests and vaccines for terrestrial animals, 7th ed. Office International des Epizooties, Paris, France, pp 1069–1082Google Scholar
- 15.Gallardo C, Nieto R, Soler A, Pelayo V, Fernández-Pinero J, Markowska-Daniel I, Pridotkas G, Nurmoja I, Granta R, Simón A, Pérez C, Martín E, Fernández-Pacheco P, Arias M (2015) Assessment of African swine fever diagnostic techniques as a response to the epidemic outbreaks in Eastern European Union countries: how to improve surveillance and control programs. J Clin Microbiol 53:2555–2565CrossRefPubMedPubMedCentralGoogle Scholar
- 16.Wilkinson PJ (2000) African swine fever. In: Manual of standards for diagnostic test and vaccines, 4th edn. Office International des Epizooties, Paris, France, pp 189–198Google Scholar