A Visual DNA Chip for Simultaneous Detection, Genotyping and Differentiation of Wild-type and Vaccine-type Classical Swine Fever Virus

Routinely, RT-PCR followed by DNA sequencing has been the method used to detect classical swine fever virus (CSFV) and exclude the interference of vaccine viruses for the field cases. Here, a DNA chip assay was developed to enable simultaneous detection, genotyping and differentiation between wild and vaccine-type CSFV. Specific oligonucleotide primers and probes were designed in the 3'non-translated region of the CSFV genome. One-step RT-PCR amplification was performed with biotin-labeled primers, followed by hybridization to the DNA probe immobilized on the plastic chip. The DNA chip can not only accurately differentiate between the three major genotypes of CSFV, but can also discriminate between wild-type and vaccine-type CSFV. The limit of detection for wild-type virus was 10 TCID50/mL for RT-PCR and 1 TCID50/mL for the DNA chip. The sensitivity of the visual DNA Chip was 10 times higher than that of the RT-PCR confirmed by agarose gel. We conclude that RT-PCR coupled with DNA probe hybridization provides a highly sensitive diagnostic tool for genotyping CSFV and discriminating between wild-type and vaccine-type CSFV in clinical samples.