Rapid detection and differentiation of wild-type and three attenuated lapinized vaccine strains of classical swine fever virus by reverse transcription polymerase chain reaction.
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. By using a one-step RT-PCR or a nested PCR followed by an agarose gel electrophoresis or a multicapillary 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 3 attenuated lapinized vaccine strains, lapinized Philippines Coronel (LPC), hog cholera lapinized virus (HCLV), and Chinese strain (C strain). The detection limit of the wild-type virus was 6.3 TCID(50) (50% tissue culture infective dose)/ml for RT-PCR and 0.63 TCID(50)/ml for nested PCR. In previous studies, notable T-rich insertions of 12-13 nucleotides (nt) were found in the 3' NTR of the genome of lapinized vaccine strains of CSFV. However, this study discovered that 2 T-rich insertions, 42 and 36 nt 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 nt length increases the size of PCR fragments, which are favorable genetic markers for rapid detection of and differentiation between wild-type and different lapinized vaccine strains of CSFV.
Pan
Chu-Hsiang
CH
Division of Hog Cholera Research, Animal Health Rsearch Institute, Council of Agriculture, Tamsui, Taipei, Taiwan.
Jong
Ming-Hwa
MH
Huang
Yu-Liang
YL
Huang
Tien-Shine
TS
Chao
Parn-Hwa
PH
Lai
Shiow-Suey
SS
D000818:Animals; D001483:Base Sequence; D006691:Classical Swine Fever / Q000175:diagnosis* / Q000821:virology; D006692:Classical Swine Fever Virus / Q000145:classification* / Q000235:genetics*; D008969:Molecular Sequence Data; D012367:RNA, Viral / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D012680:Sensitivity and Specificity; D013552:Swine; D013997:Time Factors; D014765:Viral Vaccines
D012367:RNA, Viral; D014765:Viral Vaccines