Establishment And Application Of Real-Time Quantitative RT-PCR (Taqman) Methods For Differentiation Of CSFV Virulent And Vaccine Strains And For Rapid Detection Of PRRSV Infection

China has a large pig industry worldwide, and the pig industry has brought great economic benefits, however, swine diseases have been affected and restricted the development of the industry. Recently, swine diseases showed the phenomena of mixed with two or more pathogens infection, the high morbidity and high mortality caused great economic losses in pig industry. In these diseases, classical swine fever (CSF) and porcine reproductive and respiratory syndrome (PRRS) have always been harmful to the pig industry.Classical swine fever, a highly contagious disease of pigs, characterized by high fever and hemorrhagic, caused by the classical swine fever virus (CSFV). Recently, the prevalence and clinical characteristics of CSF has changed greatly, with mild, atypical and infected sows syndrome, clinical manifestations become more complicated. In our country, the main measure to control CSF is through vaccine, but the phenomenon of vaccine failures has occurred. The large-scale application of C-strain vaccine makes it difficult to differentiate the animals infected with wild-type virus from C-strain vaccine vaccinated ones. Therefore, to prevent the spread of CSF, we must make an accurate and rapid differential diagnosis of wild virus infection and vaccination.Porcine reproductive and respiratory syndrome, a highly contagious disease of pigs, characterized by sow reproductive disorders, piglets and growing pigs respiratory disease, caused by porcine reproductive and respiratory syndrome virus (PRRSV). PRRS has occurred throughout the world, which is an important disease in pig industry. The research about PRRS has been going on around the world, but the disease has not been fully effective control measures. In 2006, the highly pathogenic PRRS outbroken in China, which exacerbated the complicatation of PRRSV, and brought significant economic losses. For PRRSV infection, we can only make the initial judgment based on clinical symptoms and pathological changes, which is difficult to diagnose. Therefore, the rapid, accurate diagnosis method for PRRS is an important measure to control PRRS.We did the research about CSF and PRRS in the following areas:1. Collected 54 CSFV strains from GenBank and compared the genomic sequences, we find an A base difference in the 5’NTTR between virulent and vaccine strains. According to the region, we designed two specific MGB probes and one pair of universal primers to identify CSFV virulent and vaccine strains. Optimized the reaction system and reaction conditions through the matrix method, we established the TaqMan-MGB fluorescence quantitative RT-PCR method to identify CSFV virulent and vaccine strains. Then we performed specificity, sensitivity and reproducibility test, established standard curve and applied the method to detect clinical samples to verify its coincidence rate compared with general RT-PCR method. The results showed that the method can specify identify CSFV virulent and vaccine strains, the sensitivity of the virulent and vaccine probes reach 101opies/μL and 102copies/μL, the method has better reproducibility, and the coincidence rate of clinical samples reaches 98.3%. Therefore, the method can be used to detect and distinguish wild virus infection from vaccination in clinical.2. Collected 30 American type PRRSV strains from GenBank, and compared the genomic sequences. We designed universal primers and TaqMan probe in the conserved region ORF6 area for American type PRRSV detection. After optimizing the reaction system and conditions, we establish TaqMan fluorescence quantitative RT-PCR method for rapid detection of PRRSV. Then we performed specificity, sensitivity and reproducibility test, established standard curve and applied the method to detect clinical samples to verify its coincidence rate compared to general RT-PCR method. The results showed that the method can specify detect PRRSV, the sensitivity reaches 103copies/μL, the method has good reproducibility, and the compliance rate reaches 95.7%. Therefore, the method can be used to rapidly detect PRRSV in clinical.In this study, the established method of identification of CSFV virulent and vaccine strains and rapid detection of PRRSV by TaqMan fluorescent quantitative RT-PCR can be applied in clinical, which provides a theoretical basis for the diagnosis and prevention of CSF and PRRS.