Simultaneous Detection Of Classical Swine Fever Virus And North American Genotype Porcine Reproductive And Respiratory Syndrome Virus Using A Duplex Real-time RT-PCR

CSF and PRRS caused by the two viruses are both among OIE listed diseases, and seriously endanger swine industries worldwide. The two diseases (especially between high fever syndrome and CSF) induce similar clinical signs (high fever, neurological, respiratory and reproductive disorders), which makes differential diagnosis of the two diseases difficult. Furthermore, co-infections with CSFV and PRRSV occur occasionally in swine herds in China and elsewhere. Design of a rapid and high throughput assay that is capable of simultaneously detecting the two agents will significantly improve the ability to identify the two viruses in swine herds rapidly.This study was aimed to develop a multiplex real-time RT-PCR for the quantitative and differential detection of CSFV and PRRSV. All genomic sequences of pestiviruses available in GenBank including 34 strains CSFV genomic sequences were aligned. CSFV specific primers and two differently labeled TaqMan probes for the differentiation of wild-type viruses from C-strain vaccine were designed in the 5′-UTR of the viral genome of CSFV. The two TaqMan probes specifically hybridize CSFV or PRRSV, respectively, in the multiplex real-time RT-PCR, with no cross-reaction to other viruses of porcine origin. Completely correct differentiation of wild-type CSFV of different subgroups (1.1, 2.1, 2.2 and 2.3 subgroups) from PRRSV (VR-2332, HuN4 and CH-1a) was achieved. There was an excellent linear relationship from 106 to 101 copies/μL when the positive standards of CSFV and PRRSV detected simultaneously in the multiplex real-time RT-PCR. The sensitivity of the assay for detecting CSFV and PRRSV was determined to be 3.2 and 1.8 TCID50 of viral RNA, respectively. Out of 155 field samples, 16 were positive for CSFV, 73 were positive for PRRSV, and 13 were co-infected with the two viruses. These results were 99.4% in agreement with those using conventional RT-PCRs. By using the assay, pigs infected with CSFV or PRRSV can be identified and removed from the swineherd, which will help to establish a CSF-free swineherd or PRRS-free swineherd. The assay provides sensitive and simultaneous detection and differentiation of CSFV and PRRSV.