The Establishment Of Monitoring Technology Platform For PRRSV And Its Preliminary Application In Early Warning System Of PRRS

ABSTRCT Porcine reproductive and respiratory syndrome(PRRS) is one of the most serious diseases affected the development of pig industry, which is caused by porcine reproductive and respiratory syndrome virus(PRRSV). PRRSV can cause severe respiratory and reproductive disorders in pigs. At present,the detected methods for PRRSV mainly are ELISA and PCR. These methods have the advantages of time saving and fast, but they are complex in operating procedures, and are prone to false positive and false negative. Therefore, it is urgent need to establish a detection techniques platform for PRRS epidemic early warning,and that is must be rapid, accurate, and high-throughput. Takes the entry-exit pigs as the object of research, we set up two kinds of real-time PCR methods on the basis of the previous, developed and reported the PCR-DHPLC method for the first time in China. To be used for monitoring and detecting of PRRS of the entry-exit pig, and to explore the establishment and application of PRRS in the early warning system, We achieved good results in the prevention and control of PRRS.3. The establishment of SYBR GreenⅠreal-time PCR detected method for PRRSVTake GP5 of PRRSV America strain as the target gene, designed primers, changed the content of each components and the cycle parameters of PCR, we eventually establish the SYBR Green real-time PCR detected method for PRRSV. The method has good specificity, and there is no cross reaction with PCV-2, PRV, CSFV; the method has good sensitivity up to 10 copies; and the method has good reproducibility, the fitting degree of the amplified curve for the same samples is high.2. The establishment of Taq Man real-time PCR detected method for PRRSVTake the N protein gene of PRRSV America strain and Nsp2 gene of of HP-PRRSV as the target gene, 2 pairs of primers and probes were designed, we established single Taq Man real-time PCR detected methods for PRRSV American strains and HP-PRRSV. We further established the double Taqman probe real-time fluorescence PCR method for detection of PRRSV and HP-PRRSV at same time on basis of single Taq Man real-time PCR. The method can differential detected PRRSV American strains and HP-PRRSV. It has good specificity, no cross reaction with PCV-2, PPV, CSFV, SIV, PRV and JEV; it has high sensitivity, 9.2 copies for PRRSV America and 56 copies for HP-PRRSV; and it has good repeatability, variation coefficient in batch is less than 1%, variation coefficient inter batch is less than 3%.4. The establishment of PCR-DHPLC detected method for PRRSVUsing GP5 gene of PRRSV standard strain as the target gene, constructed a PCR-DHPLC method for PRRSV by designing two pairs of primer for cloning and PCR-DHPLC detection. The method is good in specificity and repeatability, and sensitivity up to 10 copies. It showed that PCR-DHPLC detection method has the same sensitivity with Taqman probe Real-time PCR to be used in clinical detection, the disease can be detected that cannot be detected by conventional RT-PCR method, and PCR-DHPLC method has better applicability than other method.4. The applications of series of detected methods for PRRS in the early warning system and the establishment of early warning system framework for PRRS of exit and entry swineBy constructing the monitoring platform for PRRS based on SYBR Green I real-time PCR method for PRRSV, single and double Taq Man real-time PCR method for PRRSV and HP-PRRSV, PCR-DHPLC method for PRRSV and ELISA, we established the draft framework PRRS early warning system for the entry-exit of pig, and applied it to the isolation field and farm, to monitor PRRS, collect epidemic information and feedback to relevant departments timely. We solved the problem in early warning system that monitoring and detecting is not comprehensive or in inappropriate measures, and it played an important role in disease prevention and control for PRRS.