Development of real-time RT-PCR using MGB fluorescence probe for differentiation of wild-type and vaccine strains of classical swine fever virus

作者：Yu, Yi-Gang; Huang, Yun; Tao, Wen-Yang; Zhang, Li; Wu, Hui; Xiao, Xing-Long; Lai, Fu-Rao; Yang, Xi-Hong

来源：Modern Food Science and Technology, 2013, 29(8): 1978-1983.

• Classical swine fever virus
• Fluorescence probes
• Inhibition ratio
• Pseudovirus
• Real time RT-PCR
• Real-time PCR method
• Reproducibilities
• Variation coefficient

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 TCID50/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.

• 出版日期2013-8
• 单位华南理工大学