Development of a novel real‐time PCR assay targeting p54 gene for rapid detection of African swine fever virus (ASFV) strains circulating in Vietnam

Abstract

African swine fever (ASF) continues to cause outbreaks throughout regions of Africa, Europe and Asia. The disease can cause severe morbidity and mortality resulting in serious economic losses. Since there is no vaccine available to control ASF, early detection is critical to contain and control the disease. The aim of this study was to develop a novel real-time PCR assay based on highly conserved ASFV gene E183L (p54). The limit of detection of the assay, VNUA-p54 real-time PCR, was 2.63 copies/reaction and 2 Log 10 HAD 50 /ml. The VNUA-p54 real-time PCR was able to detect fifteen different ASFV reference strains representing p72 genotypes I, II and V. The assay was specific and did not amplify other swine viruses including CSFV, FMDV, PRRSV and PEDV. The diagnostic sensitivity of the real-time PCR assay was evaluated using 200 field clinical specimens collected from swine farms located in different provinces in Vietnam. The VNUA-p54 real-time PCR assay is an additional tool for ASF diagnostics and can be used in combination with other p72 based ASFV real-time PCR assays as a rapid confirmatory assay.

The Development of a Multiplex Real-Time Quantitative PCR Assay for the Differential Detection of the Wild-Type Strain and the MGF505-2R, EP402R and I177L Gene-Deleted Strain of the African Swine Fever Virus

“…Currently, several real-time qPCR assays for the detection of ASFV have been reported. Single real-time qPCR assays have been developed for the detection of the wild-type ASFV strains [ 29 , 30 , 31 , 32 ] and of the MGF-360-12L, UK or I177L gene-deleted ASFV strains [ 33 ]. Multiplex real-time qPCR assays have been developed for the detection of the wild-type ASFV strains [ 34 , 35 ] and of the gene-deleted ASFV strains which lacked the MGF505-2R gene [ 36 ], the MGF505-2R and EP402R genes [ 37 ].…”

Development of a multiplex qRT-PCR assay for detection of classical swine fever virus, African swine fever virus, and Erysipelothrix rhusiopathiae

“…Most multiplex qRT-PCR assays are based on target-specific TaqMan probes, which have higher specificity than conventional RT-PCR, and it is widely used for pathogen diagnosis and monitoring ( 9 ). Currently, several qRT-PCR and qPCR assays have been established for the detection of CSFV ( 10 , 11 ), ASFV ( 12 , 13 ), E. rhusiopathiae ( 14 , 15 ), and CSFV+ASFV ( 16 , 17 ). However, a multiplex qRT–PCR assay that can simultaneously and differentially detect CSFV, ASFV, and E. rhusiopathiae has not been reported.…”

Development of a highly sensitive point‐of‐care test for African swine fever that combines EZ‐Fast DNA extraction with LAMP detection: Evaluation using naturally infected swine whole blood samples from Vietnam

“…However, in many countries, animal quarantine stations and regional livestock health centres, which are responsible for waterfront quarantine, do not have sufficient diagnostic facilities. Highly sensitive and rapid real‐time polymerase chain reaction (PCR) assays are widely used for routine diagnosis of ASF (Fernández‐Pinero et al., 2013 ; King et al., 2003 ; Trinh et al., 2021 ; Wang et al., 2020 ). However, due to the significant associated cost of equipment and reagents, these methods are impracticable for use in less equipped laboratories.…”