Development of Real-time RT-PCR using MGB Fluorescence Probe for Differentiation of Wild-type and Vaccine Strains of Classical Swine Fever Virus
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Abstract:
In order to the develop multiples real-time PCR for detection and differentiation of wild-type and vaccine strains of classical swine fever virus, a pair of common primers and two differently-labeled specific MGB Fluorescence probes were designed against conserved domain of the 5’-UTR of wild-type viruses and C-strain vaccine. Moreover, in order to prevent the false negative result, pseudovirus was prepared for monitoring. The multiples real-time PCR method showed 100% specificity for the selected panel with the sensitivity being of 1 TCID50/mL for wild-type virus and 0.1 TCID 50/mL for C-strain vaccine. The reproducibility experiments showed that the variation coefficients (%CV) of intra-assay and inter-assay ranged from 0.7% to 2.2%. By using this method, CSFV RNA was detected in specimens of meat, spleen and blood with positive rates of 66.7%, 60.0% and 77.8%，respectively, while the positive rates obtained by using the standard assay was 52.4%, 40.0% and 50.0%, respectively. The result statistics showed the existence of PCR inhibitos in the clinical specimens and specimens with inhibition ratio being of 5.6~10.0%, indicating the importance of pseudovirus as IAC when PCR was used to detect the RNA of wild-type viruses and C-strain vaccine.
