Development Of A QPCR For Discrimination Of Classical And Variant Porcine Epidemic Diarrhea Virus And Analysis Of Hypervariable Region Of S Gene

Porcine epidemic diarrhea(PED)is an intestinal infectious disease caused by porcine epidemic diarrhea virus(PEDV).The disease is characterized by severe diarrhea,vomiting and dehydration.In 1971,the first reported PED was caused the economic losses of the swine industry in the world.In 2010,a new type of PEDV is widely popular in China.The variant strain has been popular around the world,and making PEDV the focus of the industry.At present,variant strains and classical strains of PEDV were popular in the swine farms,which make it difficult to detect virus and prevent and control of the disease.Based on the technical advantages of fluorescence quantitative PCR,we established the duplex TaqMan fluorescence quantitative PCR method to detect classical and variant strains of PEDV,and we also analyzed the sequence alignment,homology,genetic evolution and antigenic epitope of hypervariable region of S gene.According to the differences between the classical and variant strain S gene,we designed a FAM probe for detecting the variant strain and a HEX probe for detecting the classical strain.At the same time,the standard curve equation of classical and variant strain were constructed,which classical strain was Ct=?3.569×lgCopynumber+40.51 and correlation coefficient(R2)was 0.998,the other was Ct=?3.58×lgCopynumber+40,correlation coefficient(R2)was 0.999.This method was strong in specificity,repeatability and stability and has a minimum detection of 10 copies/?L.When Ct value was less than 35,negative control without amplification showed positive PEDV,and according to the type of probe could determine the type of the strain.A total of 183 diarrhea disease materials collected from Jilin provinces were tested by this method,which was showed that 121 samples were positive and the detection rate was 66.12%,102 variant strains and 19 classical strains.The results showed that the variant PEDV was the current dominant strains in Jilin Province.In order to explore the difference between classical and variant strain of PEDV,we selected 10 samples from the positive samples to carry out S gene hypervariable region amplification,sequencing analysis,the sequence was imported DNAMAN software comparatively analysis with representatives of classic and variant strains at the same time.The results showed that 3 insertions and 1 deletion between classical and variant strains,which accounted for 15 base insertion and 6 base deletion with a large number of point mutations.The variant strain had 83 nucleotide changes in the SR region,accounting for 10.6%(83/783)of the SR nucleotide region,a total of 31 point mutation C?T and T?C and mutation rate 37.34%(31/83).Sequence of the 10 samples homology analysis showed that CH-LY?CH-BC and CH-TH nucleotide sequence homology with classical were 97%~99.1%,and CH-JLJMF?CH-JLYJ?CH-SY?CH-SL?CH-DH?CH-CCGZL and CH-CCNA with variant strains homology were 97.4%~99.1%.However,the homology between the two strains was 84.9% ~88.3%,which indicated that the variation of the hypervariable region were significantly.Genetic evolution analysis showed that PEDV was divided into G1 clusters(classical strains)and G2 clusters(variant strain).CH-BC,CH-LY and CH-TH were divided into G1 group,which were closely related to the classical strain CV777.The others were divided into G2 clusters,which were closely related to the variant strain.Homology and genetic evolution results confirmed that the mutation of PEDV S gene hypervariable region could represent the entire S gene mutation.The comparison of antigenic epitopes of variant and classical strains proved that occurred a significant change in the 50aa~300aa.In this study,establishment of the duplex TaqMan fluorescence quantitative PCR method provided accurate and sensitive means for PEDV clinical testing,which demonstrated that the complex situation of PEDV in Jilin province,and the variation of the PEDV S gene lead to dilemma of the immune failure,it would enrich the data molecular epidemiology of PEDV and provide basic information of variant PEDV research.