The Establishment And Application Of The Dual Per Method For Senecavirus A And Encephalomyocarditis Virus

Senecavirus A(SVA)is a single-stranded RNA virus belonging to the small RNA virus family.The virus was first discovered in 2002 and was classified as a new virus genus-Seneca virus.Sows naturally infected with SVA show anorexia,transient low fever,blister fluid or ruptured blisters on the hoof and muzzle.And SVA virus infection may also cause the death of newborn piglets.Encephalomyocarditis virus(EMCV)is also a single-stranded RNA virus of the small RNA virus family.It is an important zoonotic virus.The main clinical features of acute infectious diseases.It can also cause subclinical infections,causing severe economic losses to the pig industry of the world.The genomes of SVA and EMCV are very similar,and the two viruses do not rule out the possibility of mixed infection in the clinic.In this study,the 3D genes of conserved regions of SVA and EMCV were selected to design synthetic primers,and the dual fluorescent quantitative PCR and dual RT-PCR methods that could simultaneously detect SVA and EMCV were established.Both of these methods can be used to monitor SVA and EMCV infection in pigs and preliminary epidemiological investigation.The specific contents of this study are as follows:1.The establishment of the dual PCR method for Seneca virus A and encephalomyocarditis virusIn this study,primers and probes were designed and synthesized based on the 3D gene sequences of SVA and EMCV.By optimizing the reaction conditions,a dual real-time PCR and a dual RT-PCR method that can simultaneously detect two viruses were established.The sensitivity,specificity and repeatability of the two detection methods were tested,and the two methods were compared.The specific test results showed that these two detection methods can diagnose SVA and EMCV,and have no amplification to other viruses,and have good specificity.The sensitivity test results showed that the minimum concentration of the dual real-time PCR to detect SVA and EMCV is 7.6×102copies/?L and 9.8×101copies/?L.The minimum concentration of SVA and EMCV detected by the dual RT-PCR is 1.0×105 copies/?L and 7.6×104copies/?L,respectively.The detection sensitivity of the dual real-time PCR method to SVA and EMCV is 1000 times that of the dual RT-PCR.The standard curve established by the dual real-time PCR method also has a good linear relationship.In the repeatability test,the variation of the dual real-time PCR method was less than 5%.And the dual RT-PCR method was used for three repeated detections of the templates of high,medium and low concentrations,and the results were stable and consistent.The above results showed that the two detection methods established in this study can be used for diagnosis of SVA and EMCV,and have good sensitivity,specificity and repeatability.Comparing the two detection methods,it could be seen that the dual real-time PCR method is more sensitive and suitable for clinical detection.2.The clinical application of two PCR methodsThe two PCR methods established in this study were used to detect 132 disease samples collected from pig farms around the country.The results showed that the dual real-time PCR detected 69 positive samples with a positive rate of 52.27%,including 67 SVA positive samples with a positive rate of 50.76%;26 EMCV positive samples with a positive rate of 19.70%;23 mixed positive infections amples with a rate of 33.33%.67 positive samples with a positive rate of 50.76%were detected by the dual RT-PCR.Among them,62 were SVA-positive disease materials with a positive rate of 46.97%;17 EMCV positive samples with a positive rate of 12.88%;12 mixed positive infection samples with a positive rate of 17.91%.The above results showed that the two established PCR methods can be used for clinical detection.The dual real-time PCR method has higher sensitivity than the dual-RT-PCR method,which can detect SVA and EMCV more effectively and has good application prospects.The positive rate results of the clinical samples indicated that SVA and EMCV infections are common in Chinese pigs,and the mixed infection rate of the two viruses is relatively high.