Establishment And Application Of TGEV-PEDV-PDCoV Gene Chip Technique

Swine enteric coronavirus is a long-term hazard to the healthy development of pig farming,mainly causing diarrhea and death of the piglets,causing huge economic losses to the pig farming industry.Transmissible gastroenteritis virus(TGEV),porcine epidemic diarrhea virus(PEDV)and porcineδcoronavirus(PDCo V)are the three most common swine coronaviruses,with similar clinical symptoms and mixed infections,making differential diagnosis more difficult,and better differential diagnosis techniques are urgently needed.Gene chip detection technology has many advantages and is particularly suitable for the simultaneous and rapid differential diagnosis of many epidemics.In this study,we used bioinformatics to analyze the Rd RP gene and design a pair of universal PCR primers for the simultaneous detection of TGEV,PEDV and PDCo V,and designed microarray detection probes based on the amplicon differential sequences of these three viruses,respectively,to establish a gene microarray technique for rapid identification of TGEV,PEDV and PDCo V,and used this technique to conduct epidemiological investigation of three Sichuan swine coronaviruses.1.Preliminary establishment of the TGEV-PEDV-PDCo V universal primer gene chip assay.By comparing the whole genome sequences of three standard strains of porcine coronavirus,the highly conserved Rd RP gene of the coronavirus was selected as the target gene,and the universal primer sequence was designed to obtain 395bp amplicon for each strain.The PCR annealing temperature of the universal primers was determined to be 59℃;probe concentrations were 20μmol for group 1 and 50μmol for group 2;hydration conditions were 37℃for 10 h or 50℃for 2 h;hybridization buffer was 6×SSC,5×Denhardt,0.5%SDS and 100 mg/m L salmon sperm DNA;hybridization conditions were 43℃for 1 h;and Cut-Off values were positive with a S/N≥4.363.The results showed that the chip could detect only these three pathogens with high specificity,with sensitivity of 10~1 copies/μL,10~1 copies/μL and 10~2 copies/μL for PDCo V,PEDV and TGEV,respectively,and the coefficient of variation of SNR was less than 10%between and within batches,with an optimal shelf life of 60 days.2.Application of the TGEV-PEDV-PDCo V universal primer gene chip assay.In this study,the epidemiological investigation of the three coronaviruses in Sichuan was carried out using the established TGEV-PEDV-PDCo V co-testing gene chip,and compared with the national standard detection method.In summary,the new TGEV-PEDV-PDCo V universal primer detection gene chip can be used for three coronavirus assays with good potential applications.