Reporton.

AHRI report No.43

Topic

A visual DNA chip for simultaneous detection, genotyping, and differentiation of wild-type and vaccine-type classical swine fever virus

Department

¹Division of Hog Cholera, Animal Health Research Institute,

²Graduate Institute of Veterinary Medicine, National Taiwan University,

Author

Chu-Hsiang Pan^1,2, Yu-We Ku¹, Ming-Hwa Jong¹, Parn-Hwa Chao¹, Shiow-Suey Lai²*

Summary

Routinely, RT-PCR followed by DNA sequencing has been the method used to detect classical swine fever virus (CSFV) and to exclude the interference of vaccine viruses in clinical samples. Here, a DNA chip assay was developed to enablesimultaneous detection, genotyping and differentiation between wild-type 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 chips. The DNA chips not only can accurately differentiate three major CSFV genotypes, but also can 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 chips. The sensitivity of the visual DNA chip was 10 times higher than that of the RT-PCR confirmed by agarose gel electrophoresis. We conclude that RT-PCR coupled with DNA probe hybridization provides a highly sensitive diagnostic tool for genotyping of CSFV and for discriminating between wild-type and vaccine-type CSFV in clinical samples.

Keyword

Classical swine fever virus; DNA chip; Probe; RT-PCR