| Classical swine fever (CSF), caused by classical swine fever virus (CSFV), is an acute, highly contagious and devastating viral disease with high morbidity and mortality. CSF is endemic in many countries in the world, causing serious losses in the pig industry worldwide. The well-known lapinized Chinese strain of CSFV, also known as C-strain, was developed in China in the mid-1950s and has been proved to be the safest and most effective vaccine in the past half a century. The C-strain vaccine is widespread used in the world and makes huge contribution to the prevention and control of the CSF. The C-strain vaccines manufactured in our country mainly include the tissue vaccine and the cultured cell vaccine, however, they have different virus titrations and immunological efficacy.In this study, in order to quantify the virus loads of the C-strain precisely, we constructed a RNA Standard through RT-PCR and in vitro transcription and developed a one step real-time PCR assay. Then, we applied the assay to quantify the virus loads of different types of vaccines from 3 different producers, and evaluated the proliferation of the C-strain in swine umbilical vein endothelial cells (SUVECs) and bovine testicular cells (BTCs). We obtained the following results:(1)A pair of primes were designed with a T7 promotor's sequence in the 5′end of the forward one, according to the 5′UTR of the CSFV genome whose accession number is AF091507 on Genbank. The target PCR product was purified and then in vitro transcribed to a single-stranded RNA with the T7 RNA Polymerase. The RNA Standard was obtained after purification and identification of the single-stranded RNA, which had high concentration and purity, as well as good stability. The RNA Standard obtained at a time could be used for a long time when stored at -70℃.(2)The one step real-time PCR assay for detecting and quantifying the lapinized Chinese strain of CSFV was developed succesfully after the optimization of a variety of conditions, such as the annealing temperature, primer's and probe's concentration, etc. The assay was highly specific for detecting CSFV and had a minimum detection limit of 10 copies/μL RNA, 10 times higher than the conventional RT-nPCR. The linear range was from 1.0×10~8 copies/μL to 1.0×10~2 copies/μL, and the correlation coefficient was 0.999. Data of the repetitive tests showed good reproducibilities and the coefficient variation of high, medium, low concentration within and between batches were 0.54%, 0.52%, 0.39% and 1.47%, 1.85%, 1.01%, respectively.(3)Data of quantifying the virus loads of several lapinized virus vaccines against CSFV showed that the virus loads of the tissue vaccines were 10~100 times higher than that of the cultured cell vaccines comparing each producers'vaccines. However, when comparing with different producers, the virus loads of tissue vaccines and cultured cell vaccines were relatively discrepant. The maximum discrepancy was up to 20.8 times among the tissue vaccines and 30.2 times among the cultured cell vaccines. However, all the vaccines tested in this study achieved the standard made by the conventional rabbit fever reaction.(4)Data of evaluating the proliferation of the C-strain proliferated in two types of cells showed that the virus loads in SUVECs were approximately 7.1 times higher than that in BTCs, which supplied an effective techniques support for the evaluating the proliferation of the C-strain proliferated in different cell lines. |
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