Development of a novel sensitive single-tube nested PCR assay for the detection of African swine fever virus

• Published in: Archives of virology 2024-05, Vol.169 (5), p.107-107, Article 107
• Main Authors: Milton, A. Arun Prince, Das, Samir, Momin, Kasanchi M., Prasad, M. C.B., Khan, Sabia, Priya, G. Bhuvana, Ghatak, Sandeep, Sen, Arnab, Baruah, K. K.
• Format: Article
• Language: English
• Subjects: African swine fever; African Swine Fever - diagnosis; African Swine Fever - virology; African Swine Fever Virus - genetics; African Swine Fever Virus - isolation & purification; Animals; Asfarviridae; Biomedical and Life Sciences; Biomedicine; DNA Primers - genetics; DNA, Viral - genetics; Food contamination; Food security; Infectious Diseases; Limit of Detection; Medical Microbiology; Original Article; Polymerase chain reaction; Polymerase Chain Reaction - methods; Sensitivity and Specificity; Swine; Viral diseases; Virology
• ISSN: 0304-8608; 1432-8798
• DOI: 10.1007/s00705-024-06037-z

African swine fever (ASF) is a highly fatal and contagious viral disease caused by African swine fever virus (ASFV). It has caused significant economic losses to the swine industry and poses a serious threat to food security worldwide. Diagnostic tests with high sensitivity are essential for the effective management of ASF. Here, we describe a single-tube nested PCR (STN-PCR) assay for the detection of ASFV in which two consecutive amplification steps are carried out within a single tube. Two pairs of primers (outer and inner) were designed to target the p72 gene of ASFV. The primer concentrations, annealing temperatures, and number of amplification cycles were optimized to ensure the consecutive utilization of outer and inner primer pairs during amplification while minimizing the likelihood of amplicon contamination. In comparison with two conventional endpoint PCR assays (one of which is recommended by the World Organization for Animal Health), the newly developed STN-PCR assay demonstrated a 100-fold improvement in the limit of detection (LOD), detecting 100 copies of ASFV genomic DNA, whereas the endpoint PCR assays could detect no fewer than 10,000 copies. The clinical performance of the STN-PCR assay was validated using 95 tissue samples suspected of being positive for ASFV, and the assay showed 100% specificity. A Cohen’s kappa value of 0.91 indicated perfect agreement between the assays. This new STN-PCR assay is a potentially valuable tool that will facilitate the control of ASF.