Investigation And Application Of Virus And Serological Detection Methods Of Porcine Epidemic Diarrhea

Porcine epidemic diarrhea virus(PEDV) belong to Coronavirus,is the etiologcal of porcine epidemic diarrhea(PED),which is a highly contagious enteric disease in pigs causing vomit,diarrhea,dehydration. The epidemiological characteristics,clinical symptoms or pathological changes of the small intestine of Porcine epidemic diarrhea (PED)are very similar with porcine transmissible gastroenteritis(TGE) and porcine rotavirus (PRV),so the detection according to clinical experience and pathological changes can only be an initial diagnosis.Currently there is no efficient way to distinguish these viruses.In recent years due to the frequently breaking out of diarrhea in pigs and the lack of effective way,the world’s pig industry had suffered huge economic losses.Therefore,the establishment of fast,accurate and simple detection methods has an important significance for PED molecular biology and clinical preservation.This study established RT-PCR and real-time quantitative PCR mothod to detect PEDV while IFA and neutralization method were established to detect PEDV antibodies.RT-PCR:full-length fragment of ORF3was amplified using the specific primers designed according to PEDV-ORF3gene sequence deposited in GenBank, the amplified fragments were connected to vector pMD18-T and transformed into TOP10competent cells and extracted plasmid,sequencing and analysis.The RT-PCR method was established by optimizing the annealing temperature to determine the best reaction system and amplification procedures.Good amplification efficiency in the55.9℃.The sensitivity of the assay was1.86pg RNA.The RT-PCR method was specific for PEDV because no amplification was found for TGEV、PRV、PRRSV、HEV and control.The positive rate of clinical samples was80%.Real-time quantitative PCR: The specific target fragments were obtained though RT-PCR amplification using specific primers designed according to the GenBank deposited PEDV M gene,then the amplified fragments were connected to vector pMD18-T and transformed into TOP10competent cells,extracted plasmid and identified them with restriction enzyme.The concentration and copies of the recombinant plasmid were measured. The quantitative PCR method was established by making the standard curve by dilutions of plasmid and got a good linear relationship,R2＝0.997and optimizing the conditions to determine determine the best reaction system and amplification procedures.The sensitivity of the assay was3.168copies/μL plasmid DNA.The assay was specific for PEDV because no amplification was found for TGEV、PRV、PRRSV、HEV and control.The coefficient variation (CV)of intra/inter-assay for the same DNA sample were less than2%. The positive rate of clinical samples was98%.IFA:24-well plates were covered by LR7cells which were adapt to the PEDV vitro culture, according to4.0-5.0×105/mL,24h,then covered with virus at MOI=0.1, at37℃ for lh.The immunofluorescence method was established by adding primary and secondary antibodies after36-48h, seting up one negative serum control, control without an primary antibody control and normal cells. The fluorescence was observed under fluorescence microscope.Results: Specific fluorescence was observed under the microscope,and the negative serum control,without an anti-virus control and normal cells control were no specific fluorescence appeared. The results indicated that the immunofluorescence were specificity.Relative quantification neutralization assay: the different dilutions of positive serum was inserted PEDV-GFP virus,neutralized30min at37℃,joined in the24-well plates covered with VERO cells.24-well plates was observed under a fluorescence microscope after12h.Then the cell was splited by cell lysates and measured fluorescence values with TBS-380Mini fluorescence.The relative quantitative neutralization assay was developed by establishment of a linear relationship to comparison of serum antibodies.Results:the fluorescence gradient was observed under fluorescence microscope afterl2h,the fluorescence value can be reflected a good level of antibody of serum. The results indicated that the establishment of neutralization assay was reliable.