Establishment Of Rapid Detection Methods For PDCoV And PEDV And Genetic Variation Analysis Of PDCoV

Coronaviruses are common throughout the world,they can infect people and animals.Gene recombination was easily occurred during the coronavirus evolution,because of its widely host ranges and the genome structural characteristics.Therefore the new subtype and new coronavirus appear constantly.Porcine deltacoronavirus(PDCo V)is a newly discovered porcine viral diarrhea virus.It mainly infects piglets and causes severe enteritis accompanied by diarrhea and vomiting and other symptoms.This symptoms are symptomatically indistinguishable from those caused by porcine epidemic diarrhoea virus(PEDV)and transmissible gastroenteritis virus(TGEV).Clinically the mixed-infection of PDCo V and PEDV is more serious which causes huge economic losses to the swine industry.In order to control and prevent effectively these diseases,the researches of rapid detection methods for PDCo V and PEDV as well as genetic variation analysis of PDCo V wild strain are the primary tasks.In current study,we first established the fluorescence quantitative RT-PCR for detecting PEDV and PDCo V respectively,then establish the dual fluorescence RT-PCR method and applied to the detection of clinical samples.At the same time,At last the whole genome sequence of PDCo V Henan strain CH-01 was determined,and the genetic variation was analyzed.The research laid a foundation for the further research on the biological characteristics and pathogenicity of PDCo V.1.A pair of primers were designed and synthesized according to the published PDCo V N gene in Gen Bank.The 260 bp fragment was successfully amplified from CH-01 PDCo V strain,and the porcine recombinant plasmid of PDCo V N gene fragment was constructed.After verified by sequence,the recombinant plasmid was 10-fold dilution,SYBR Green I fluorescence quantitative RT-PCR was carried out,and reaction system and the reaction conditions were optimized.The system automatic analysis software showed that there was a good linear relationship between the Cq value and logarithm of recombinant plasmid.Kinetic curve analysis showed that the sensitivity of the method is 54 copies/?L.The specificity tests showed no cross-reactivity with TGEV and porcine pseudorabies virus(PRV),porcine circovirus type II(PCV2),PEDV,porcine reproductive and respiratory comprehensive syndrome virus(PRRSV).The establishment of PDCo V fluorescence quantitative RT-PCR method laid the foundation for detecting and monitoring of PDCo V infection in clinic.2.A pair of specific primers for N gene were designed and synthesized according to the reported PEDV N genome in Gen Bank.The N gene was successfully amplified and subcloned into p MD18-T vector.After 10 times gradient dilution of the recombinant plasmid,SYBR Green I fluorescence quantitative RT-PCR was carried out,and reaction system and the reaction conditions were optimized.Analysis software automatically showed that there is a good linear relationship between the Cq value and the logarithm of the recombinant standard quality grain,Correlation coefficient R2 is 0.99.Kinetic curve analysis showed that under the optimized reaction conditions,The minimum detection limit is 57 copies /?L by this method and TGEV,PCV2,PRV,PRRSV and PPV could not be detected.So the method showed good specificity.We amplified N gene for three times using the previous fluorescent quantitative RT-PCR amplification method.The results showed that variation coefficient was less than 1% between inter-and intra-group.It can be used for quantitative and qualitative detection of PEDV.3.On the basis of the establishment of the single SYBR Green I fluorescence quantitative RT-PCR of PDCo V and PEDV,the double SYBR Green I fluorescence RT-PCR method of PDCo V and PEDV was established successfully through optimizing the amplification conditions.That is both PDCo V and PEDV can be detected at the same time by double SYBR Green I fluorescence RT-PCR method.The analysis of sensitivity,specificity and reproducibility of the detection methods showed that the detectable limit was 28.6 copies/?L for PDCo V and 99.6 copies/?L for PEDV.And TGEV,PRRSV,PRV,PCV2 was negative when using this method.It provided technical support for the rapid diagnosis of the virus.4.In order to understand the genetic variation of PDCo V in Henan Province,in China,13 pairs of specific primers with overlapping of PDCo V were designed and synthesized according to the published PDCo V genome in Gen Bank.The whole genome of PDCo V Henan strain CH-01 was sequenced and analyzed.Results showed that Henan CH-01 total includes 25 403 bp.The coding gene sequence is 5'-ORF1a/b-S-E-M-NS6-N-NS7-3'.The PDCo V CH-01 stain examined in this study shared 97.3% to 98.1% nucleotide identities with with the other PDCo V strains available in Gen Bank,and shared 97.3% to 98.1% amino acid identities with other PDCo V strain.The nucleotide identities of ORF1 gene with other domestic and foreign strains is among 98.4%~98.8%,the nucleotide homology of S gene with other strains is among 98.7%~99.1%.Phylogenetic trees were constructed by using the whole PDCo V genome,entire S,ORF1,N,M and E gene sequences.The whole PDCo V genome and ORF1 trees showed that all PDCo V were clusted in a large branch,which showed that the current domestic and international popular PDCo V was in one type.Further analysis indicated that all the China strains clustered into a subclade with the PDCo V strain CH-01,while PDCo V strains isolated in other countries were clustered separately.The establishment of single fluorescence quantitative RT-PCR and dual fluorescence RT-PCR for detection of PEDV and PDCo V provides an effective molecular biology diagnosis method for these two diseases differential diagnosis and mixed infection.The analysis of the complete nucleotide sequencing of the strains in Henan PDCo V provides theoretical basis for further researching and analyzing biological characteristics of PDCo V,gene structure and protein function.