Development and application of real-time TaqMan RT-PCR assay for improved detection of classical swine fever virus in slaughtered pigs

Abstract

Classical swine fever (CSF) is an economically devastating disease of pigs. Instrumental to the control of CSF is a wellcharacterized sensitive assay that can deliver a rapid and accurate diagnosis before the onset of clinical signs. With this objective, a real-time fluorogenic-probe hydrolysis (TaqMan) reverse transcription-polymerase chain reaction (RT-PCR) assay was developed for rapid and specific detection of classical swine fever virus (CSFV) and applied on samples derived from infected slaughtered pigs. A pair of PCR primers targeting 5' -non-coding region (CSFL1 and CSFR1) in conjunction with a CSFV-specific fluorogenic probe (CSFP1) was designed and assessed in real-time PCR. During PCR, when the target of interest was present, the CSFV specific FAM-labeled TaqMan probe annealed to the amplicon between the forward and reverse primers and was subsequently cleaved via the 5-3 exonuclease activity of the DNA polymerase resulting in the release of the fluorescent reporter dye. This assay was found to be rapid and strain-specific for CSFV detection.

Transmission of African swine fever virus to the wild boars of Northeast India

“…For detection of RNA viruses, the extracted RNA was converted into cDNA by reverse transcription using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA) as per the manufacturer’s instruction. The samples were screened for CSFV by Real-Time PCR as described earlier (Rout and Saikumar 2016 ). Conventional RT-PCR was used to screen the samples for PRRSV as per OIE guidelines (Wernike et al.…”