Differential Detection Of Classical Swine Fever Virus Using MGB Real-Time PCR Method And The Investigation Of It's Dynamic Distribution

Classical swine fever(CSF),caused by the Classical swine fever virus(CSFV),is a highly contagious and often fatal disease of swine.A real-time RT-PCR assay using TaqMan minor-groove-binding(MGB) probe was developed and evaluated to discriminate wildtype stains and C-strain of CSFV.We finished the primary assemble for the detection kit. Meanwhile,a relative quantitative real-time RT-PCR assay usingβ-actin as an inner reference was developed to determine the dynamic distribution and tropism of the highly virulent SM strain in acutely infected pigs.In order to investigate the replication,dynamic distribution and tropism of the CSFV in acutely infected pigs and to provide technical support for the differential diagnosis of CSFV,a rapid relative quantitative system is developed combining real time fluorogenetic quantitative PCR technology(FQ-PCR) and the ABI7500 detection system.One set of primers and TaqMan-MGB probe was designed based on the sequence of CSFV,BVDV and BDV genomic sequences from the GenBank.While, another set of primers and TaqMan probe was designed based on the reference geneβ-actin (ACTB) sequence to validate the FQ-PCR.The CSFV and ACTB amplification system were optimized and excellent amplification efficiency was obtained in a high level of 91.7%and 92.4%respectively.The linear correlation coefficient from the standard curve was both 0.998.Better specificity was demonstrated by the negative result of such as BVDV,PRRSV and FMDV amplification.What's more,the method was evaluated to discriminate wild-type stains from wild-type stains and Hog Cholera Lapinized Virus(HCLV) strain,which could avoid the interference from HCLV.6 batches of HCLV vaccine samples were used to validate the method.Sensitivity analysis show an order of magnitudes higher then the housekeeping RT-nPCR method.The coincidence rate was 94.3%in detecting 122 clinical samples compared with the RT-nPCR(115/122).Sixteen of the pigs Changbai comprised the experimental group were given an iUTRamuscular injection with 1 ml of blood containing4×10^3.84TCID₅₀(50%tissue culture infection dose) SM virus.Clinical signs of CSF were evaluated by using a score system suggested previously in order to follow the progression of the disease.Body temperature was recorded daily.Two experimental pigs were killed randomly every day between days 1 and 8.Twenty-two tissues and organs were collected from each pig and were analyzed quantifiably by the FQ-PCR using 2^{-△△CT} model.The control group comprised one pig,which was injected with the same volume of PBS.Blood and tonsil samples were collected daily until day 8 when the pig was killed.Twenty-two types of tissue and organs were also collected as described for the experimental group.The samples were tested for CSFV and were all antigens negative.As the data shown:1.Viral RNA could be detected in all the samples of the infected pigs from day 1 post infection and reached a peak at day 8 when the pigs were close to death.2.The viral load progressively increased over the 8 days.The virus replicate fastest in ileum,liver and submandibular lymph nodes with 5.997,5.649 and 5.175 order of magnitude respectively compared to that of 1DPI While,the virus replicate most slowly in kidney, and muscle with 1.432 and 0.797 order of magnitude respectively compared to that of 1DPI 3.The general trend of tissue tropism for the 22 tissue samples taken from each of the 16 pigs according to the viral load(from high to low) was as follows:pancreas, mesenteric lymph node,inguinal lymph node,ileum,submandibular lymph node, spleen,jejunum,skin,lung,liver,tonsil,rectum,ileocaecal valve,kidney,spinal cord, esophagus,bladder,stomach,duodenum,muscle,brain and heart.The clinical score of the infected pigs showed a continued increasing trend from day 1 to day 8 post infection,which was found to correlate well with the virus load ratio. The noninfected control kept at a score of 0.The body temperature began to increase on the 2th day after infection.Then the temperature was kept around continued 41.0℃for 1.5 to 2 days,then began to decline at day 4 to 5 post infection.However,it showed irregular changes from day 6 to day 8 near to death.While the body temperature of the control group maintained at the normal level of 39.5℃.Specific immunoreactivity was observed in all tissues and organs tested and the positive signal showed an increasing trend similar to the result of quantitative detection previously described from day 1 to day 8 post infection.The positive signals appeared firstly in capillaries and then gradually transfer into the lymphocyte with an increasing trend.While the control group showed no positive signal.The developed CSFV TaqMan MGB-PCR differential detection method has offered strong technical support for the eradication of CSF.Meanwhile,it lay a foundation for the further research of pathogenic characteristic and pathogenesis.