Validation of a real-time PCR assay for the detection of African swine fever virus in fresh pork meat juice

Abstract

African swine fever virus (ASFV) is the etiological agent of African swine fever (ASF), a disease with detrimental effects on the health, welfare, and production of domestic and wild pigs. The ASF laboratory confirmation is based on the analysis of blood, serum and organ samples. However, testing these samples could not be always convenient, economically feasible or possible. This study describes the validation process of a PCR-based assay targeting a portion of p72 gene, used for the molecular detection of ASFV, from meat juice samples obtained from pigs succumbed to ASFV. More specifically, we investigated the capability of a real-time PCR assay to detect ASFV DNA in meat juices obtained from the diaphragmatic muscle along with the correspondent spleens of 55 ASFV-positive pigs and wild boars sampled from confirmed outbreaks in Romania and from 73 ASFV-negative and regularly slaughtered healthy pigs collected in the Abruzzo region (Italy). The test was able to detect viral DNA in both types of samples, with lower Ct values in spleens (mean=21.11, median=20.61) than meat juices (mean=23.08, median=22.40). However, distributions of Ct values were strongly correlated each other (R²= 0.83, P<0.001). Considering the distribution of the observed Ct values in the 55 positive meat juice samples, a 1:10 dilution would be able to detect 90 % of positive samples, whereas a 1:100 dilution would reduce the detectability to 78 % of more contaminated samples. As meat juice could be obtained easily from muscles and considering the potential use of this test on pooled samples, it could represent a tool to aid the investigation of ASFV spread.