A one-step, gel-based RT-PCR assay with comparable performance to real-time RT-PCR for detection of classical swine fever virus.

Classical swine fever, a notifiable disease to the Office International des Epizooties (OIE), is a highly contagious viral disease affecting both domestic pigs and wild boars. Rapid, sensitive, and specific detection of the causing agent classical swine fever virus (CSFV) is therefore essential for diagnosis and control of the disease. Most protocols for gel-based PCR consist of two steps, reverse transcription followed by PCR. Such a protocol is time consuming, laborious and more prone to contamination. Two highly sensitive and fast one-step RT-PCR assays were developed for gel-based and real-time detection of CSFV, and their performances were compared to that of a published real-time assay. The results showed that the gel-based assay had comparable performance to the real-time RT-PCR assays for detection of the virus. A detection limit of 50 copies was achieved by both assays. It is concluded that the one-step gel-based RT-PCR assay provides the simplest and most sensitive method for detection of CSFV in cell culture material or clinical samples, that can be applied in laboratories without facilities for real time PCR assays. The one-step format minimizes the risk for cross contamination and the hands-on time. The real-time assay is suitable for high-throughput screening of the virus in large populations.