| Transmissible gastroenteritis virus (TGEV) of swine mainly cause porcine gastroenteritis infection.TGE is a highly contact of communicable disease and it can generally cause such as vomiting, diarrhea, dehydration, thin and piglet death, especially for about two weeks of piglet that the mortality rate could reach 100%. It mainly breaks out in winter and early spring which disseminated quickly, cases livestock breeding a huge loss. The laboratory stressed major standardisation of RT-PCR methodology is used for detecting TGEV, comparing fluorescent quantitation RT-PCR methodology, sensitivity is lower. Conventional RT-PCR is applicable to the serious symptom of pigs, but for invisible infection or early infection,it need sensitivity higher detection method to implement so it can take effective measures to control validly in reducing TGE outbreak of the losses in the early infection.This experiment is mainly for clinical tests to establish a higher sensitivity Taqman real-time fluorescent quantitation RT-PCR methodology. By highly conservative sequence of N protein gene the TGEV comparing with genbank has published sequence, selecting a sequence of a small area, design and probe and through out a positive standard of the clone preparation, by the standard curve illustrate that concentration of reaction templates and the fluorescent quantitative CT value has a good linearity relation. The equation of a straight line for y=-3.15181gx+51.613. The correlation coefficient achieves 0.997 and the method has a high level of sensitivity and good Specificity. It can be detected at the bottom of the TGEV DNA which the content is 1.445×10-6 ng/μl. At the same time compared with conventional RT-PCR method, sensitivity is 100 times higher than conventional's. Because the detection methods is more sensitive than conventional RT-PCR way, the early infection of pigs can make a better accurate judgement, which can prevent and control the outbreak of TGE can play an important role. Meanwhile, according to the prevention, currently clinical TGE mainly adopts the vaccine immune to undertake, other drugs can only improve the body's resistance and prevent the auxiliary function secondary infection. So vaccination immune to TGE prevention and treatment is particularly important.The lab uses salmonella as the carrier to construct a pig contagion enterogastrtisetc genetic engineering vaccine and make animal immune protective test. A total of 42 TGEV negative pigs were used, which divided into four groups:blank tapping poison group, empty carrier tapping poison group. immune artificial infection group and control group. First through the immune infection, and then artificially immune protective test. Clinical symptoms reveals that blank group and empty carner artificial infection group show serious TGE symptoms, however, immune artificial infection group of clinical symptoms are not obvious. After HE it can discover that blank group tapping poison and empty carrier artificial infection group has a severe pathological changes, its small intestinal villi has a serious necrosis and fall off, and application immunohistochemical and fluorescence quantitative RT-polymerase chain reaction (RT-PCR) method found that the virus detect chiefly in the small intestine, which proved that after TGEV infection it mainly appear diarrhea, dehydration and thin symptoms. Through the animal experiments,we can reveal that the genetic engineering vaccine has a good protection at piglets. At the same time through quantitative fluorescence detection of each organization, we can conclude the distributing of TGEV situation in the body of pigs. |
PDF Full Text Request