Establishment and Application of a Real-time Fluorescent Quantitative PCR Method for Detection of Porcine Circovirus Type 2

作     者：Dong Lin Wei Feng Guan Yu Liu Zengshan Shen Zhiqiang 

作者机构：College of Animal Science and Veterinary MedicineJilin University Shandong Binzhou Animal Science and Veterinary Medicine Academy 

出 版 物：《Animal Husbandry and Feed Science》 (动物与饲料科学（英文版）)

年 卷 期：2015年第7卷第4期

页      面：249-252,256页

学科分类：090601[农学-基础兽医学] 09[农学] 0906[农学-兽医学] 

基　　金：Supported by Shandong Province Natural Science Fund Project 

主　　题：Porcine circovirus type 2 Fluorescent quantitative PCR SYBR Green I 

摘      要：[ Objective ] To establish a real-time fluorescent quantitative polymerase chain reaction （PCR） method with SYBR Green I for the detection of porcine circovirus type 2 （PCV2）. [Methods] Specific primers were designed to amplify the conserved gene segments of PCV2 with a size of 177 bp by PCR. The ampli- fied gene was cloned into the vector of pMD 18-T and transformed into DHSct to screen positive clones. After being extracted and purified, the recombinant plasraids pMD 18-T-177 were taken as the standard DNA templates to establish the fluorescence quantitative PCR method for the detection of PCV2, and the PCR re- action conditions were optimized. [ Results] Ct value of the established PCR method showed a good linear relationship with the standard DNA templates within a viral load of 3.21 × 100 -4.16 × 108 copies/μL , the correlation coefficient was O. 998 8 and the slope was - 3.286. The method did not show any cress-reactions with the genomes of PRRSV, PCV1, CSFV, PRV, PPV and Escherichia coli. Sensitivity of this method was proved to be 3.21 × 10 copies/μL, which was 1 000 times higher as conventional PCR method. Variation coefficients of the repeated trims among same batch or different batches were both less than 3.00%. Positive rate of clinical samples detected by the established PCR method was 58.94%, which was significantly higher than the detection rate by conventional PCR. [ Conclusions ] A reM-time fluorescent quantitative PCR method with SYBR Green I for the detection of PCV2 was established, which was better for conducting the quan- titative analysis and the early diagnosis of PCV2 infection.