Abstract:

【Objective】 A rapid one step fluorescent quantitative PCR assay using TaqMan minor-groove-binding (MGB) probe was developed and evaluated to discriminate wild-type stains from wild-type stains and hog cholera lapinized virus (HCLV) strain of classical swine fever virus (CSFV). 【Method】 A pair of universal primer and a wild-type specific MGB probe were designed from the conservative region of CSFV genome. Then the method was optimized. Specificity, sensitivity, and conformity were tested. 【Results】 The detection results of 24 quality-control samples showed good specificity. The sensitivity of the assay was 5.3×10-2pg viral RNA per reaction, and showed a good linear relationship at a range of 100-10-7. The coincidence rate was 94.3% in detecting 122 clinical samples compared with RT-nPCR (115/122),and the positive coincidence rate and negative coincidence rate were 86.4% (38/44) and 98.7% (77/78), respectively, Kappa value was 0.87＞0.75. 【Conclusion】 The one-step TaqMan-MGB PCR is able to differentiate wild-type and HCLV of CSFV strain, which shows good specificity, sensitivity and also shows high consistency of TaqMan-MGB Probe Technology and RT-nPCR method. This assay may serve as a useful diagnostic tool for CSF.

Key words: classical swine fever, fluorescent quantitative PCR, TaqMan-MGB, CSFV