Establishment Of Taqman Probes Fluorescence-Based Quantitative Real-time PCR For PCV2 And Protective Effect Of PCV2 Vaccine On Balb/c Mice

Porcine circovirus type 2(PCV2)can cause the dysfunction of swine immune system and porcine circovirus-related diseases(PCVAD)such as weaned pig multi-system failure syndrome(PMWS),and has become one of the most important pathogens in pig farming.The biological characteristics of the pathogen,the pathogenic mechanism and the vaccine protection efficacy can be conveniently studied by means of establishing an ideal xenogeneic animal and this animal model.Many literatures have shown that virus replication,seroconversion,and microscopic lesions could be found in PCV2 vaccinated Balb/c mice.Mouse animal models may provide great convenience for antiviral agent and vaccine research.The TaqMan probes fluorescence-based quantitative real-time PCR detection technology established in this study was aims to provide a reliable tool for the quantitative detection of PCV2 virus nucleic acid content,and to study the PCV2-related diseases and the evaluation of the effectiveness of PCV2 vaccines.Based on the conserved gene sequence of PCV2 ORF2 in NCBI,primers and probes were designed and synthesized.By optimizing the reaction conditions and reaction system,a fluorescence-based quantitative real-time PCR detection method for specific quantitative detection of PCV2 was successfully established.The results showed a good linear relationship when the sample concentration is between 1.9×10~3?1.9×10~9 copies/?L.The correlation coefficient reaches 0.999.The detection method has high sensitivity,which is 1000 times more sensitive than ordinary PCR,and the minimum detection concentration is 101 copies/p,L.The established method also has good specificity and repeatability.The TaqMan probes fluorescence-based quantitative real-time PCR detection method established in this study is consistent with the national standard qPCR method and can be used as a new method for quantitative detection of PCV2.Based on the established TaqMan probes fluorescence-based quantitative real-time PCR detection method,52 SPF-grade Balb/c mice were selected for PCV2 vaccine immune and challenge test,in order to experimental reference of PCV2 pathogenicity and the vaccine evaluation research.The tested mice were randomly divided into 3 groups,control group,PCV2 challenge group,immune and challenge group respectively.At day 3,7,14 and 21 after PCV2 challenged,the physical characteristics,mental status,and abnormal lesions of the tested mice were detected during experiment.And then the viral load in the heart,spleen,liver,lung and kidney were tested.The results showed that the mice in the challenged control group were significantly thinner among those in the other two groups.The virus could be tested in multiple organ tissues of mice at the 3rd day after challenged.The order of organ virus content from more to less was:liver,spleen,kidney,lung,and heart.The virus load of PCV2 in the organ tissues of mice in the immuned and challenged group was lower than that in the challenged group.Except for the lungs,the virus load in the other organs showed a significant decrease.It indicated that Balb/c mice can be used as xenobiotics to study PCV2 immune and challenge protection,and PCV2 vaccine can provide certain protection to mice.