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Rapid detection and differentiation of wild-type and attenuated lapinized vaccin...  

Update Date: [2013-11-14]
No.:43

Reporton.
AHRI report No.43
Topic
Rapid detection and differentiation of wild-type and attenuated lapinized vaccine strains of classical swine fever virus by reverse-transcription polymerase chain reaction
Department
1 Division of Hog Cholera Research, Animal Health Research Institute, Council of Agriculture, Executive Yuan,
2 Graduate Institute of Veterinary Medicine, National Taiwan University,
Author
Chu-Hsiang Pan1,2, Ming-Hwa Jong1, Yu-Liang Huang1, Tien-Shine Huang1, Parn-Hwa Chao1, Shiow-Suey Lai2,*
Summary
A simple one-step reverse transcription-polymerase chain reaction (RT-PCR) method was developed based on T-rich insertions in the viral genome for simultaneous detection and differentiation of wild-type and vaccine strains of classical swine fever virus (CSFV). The CSFV-specific primers were designed to contain the sequences of the T-rich insertion sites that exist uniquely in the 3' nontranslated regions (3'NTR) of the genome of lapinized CSFV vaccine strains. Using a one-step RT-PCR or a semi-nested RT-PCRfollowed by an agarose gel electrophoresis or a multi-capillary electrophoresis, the wild-type and lapinized vaccine strains of CSFV in clinical samples could be detected and accurately distinguished. These assays can be applied to at least three attenuated lapinized vaccine strains, LPC (lapinized Philippines Coronel), HCLV (hog cholera lapinized virus), and C (Chinese)-strain. The detection limit of the wild-type virus was 6.3 TCID50 (50% tissue culture infective dose)/ml for RT-PCR and 0.63 TCID50/ml for semi-nested RT-PCR. In previous studies, notable T-rich insertions of 12–13 nucleotides (nts) were found in the 3' NTR of the genome of lapinized vaccine strains of CSFV. However, this study discovered that two T-rich insertions, 42 and 36 nts in length, are present in the viral genome of lapinized vaccine strains LPC/PRK (primary rabbit kidney) and LPC/TS (Tam-Sui), respectively. These T-rich insertions of 12, 36, and 42 nts length increases the size of PCR fragments, which are favorable genetic markers for rapid detection and differentiation of wild-type and different lapinized vaccine strains of CSFV.
Keyword
Classical swine fever virus; lapinized vaccine strains; nontranslated region; reverse transcription polymerase chain reaction.