Development And Preliminary Application Of Fluorogenic Quantitative RT-PCR For Detection Of Hog Cholera Lapinized Virus

Classical swine fever(CSF),caused by Classical swine fever virus(CSFV),is a highly Contagious disease of pigs,and one of OIE listed diseases.In China, CSF is still one of the most severe disease.Now both hog cholera lapinized virus (HCLV) rabbit spleen and lymph tissue original vaccine and tissue culture origin were spreadly used to prevent pigs from CSF in pig farming of all the world. There is no Cytopathic Effee, caused by HCLV. The vaccine quantitative result has to be detected by rabbit fever reaction.But this method can not detect the quantity of HCLV exactly,the period of the exam is always long and the results are usually influenced by rabbit.So how to detecte the vaccine quantitative result is a problem.A fluorogenic-probe hydrolysis (Taqman)-reverse transcriptase PCR assay was developed for the quantitative of Hog cholera lapinized virus (HCLV) vaccine. The method contains a pair of primers and an internal fluorogenic probe designed to target a region within the E0 of the HCLV genome. The sensitivity of the assay was 3.84 copies/μL.No positive was observed,taking Porcine parvovirus virus (PPV), Pseudorabies virus (PRV), Porcine reproductive and respiratory syndrome virus (PRRSV), Bovine viral diarrhea virus(BVDV) as templates.The coefficient of variation(CV) of 3 HCLV cDNA samples is 1.05%～1.84% intra-assay and 3.70%～5.43% inter-assay.32 cell cultures,9 vaccines(tissue culture origin)and183 vaccines(spleen and lymph origin)were quantified by this method and give similar but more accurate results compared to the conventional rabbit fever reaction(rabbit infection dose,RID).By using this assay, the quality of Classical Swine Fever Virus of 24 different periods in primary bovine testicular cells were detected. The study showed that virus loads increased with the time and at the 96th hour the titre of progeny virions was the most.In conclusion, the method established can guide how to confect the vaccine and determine which time to gather the cell cultures,which has the advantages of high sensibility, specificity and good stability.