Establishment And Preliminary Application Of Quadruple RT-PCR Detection Method For Four Diarrhea Viruses In Pigs

Pig diarrhea-associated viruses severely restrict the healthy growth of the herd,causing unpredictable losses in the pig industry,including porcine epidemic diarrhea virus(PEDV)and porcine transmissible gastroenteritis virus(TGEV),pig A group rotavirus(PoRV)and newly discovered pig delta coronavirus(PDCoV)are more common around the world.At present,the identification of these four diarrhea viruses is mainly based on single RT-PCR technology.Although the technology has certain specificity and convenience,when it is targeted at large-scale epidemiological investigations,it exposes long time and fees.The disadvantages of manpower.This study established a quadruple RT-PCR detection method that can simultaneously identify four viruses(PEDV,TGEV,PoRV,PDCoV).After preliminary laboratory clinical application,it proved that the detection method has high reliability and sensitivity.And the specificity is good.The gene sequences of PEDV,TGEV,PoRV and PDCoV were downloaded by NCBI,and four pairs of specific detection primers were designed by DNAMAN,DNAStar,MEGA6 and other biological software.The annealing temperature and enzyme concentration were optimized respectively.The specificity of the four single RT-PCR detection systems was verified by using the optimized four single RT-PCR detection systems,which proved that the four single DNA detection methods had good specificity.The recombinant plasmids of four viruses were constructed.the results showed that the detection limits of PEDV,TGEV,PoRV and PDCoV were1.1×103、9.8×102、0.11×103、1.26×103copies/μL,respectively.In this study,a single RT-PCR detection system for PEDV,TGEV,PoRV and PDCoV was established.on the basis of this study,the plasmids of the four viruses were used as templates.Six kinds of double RT-PCR detection systems,PEDV\PoRV,PEDV\TGEV,PEDV\PDCoV,PoRV\TGEV,PoRV\PDCoV and PDCoV\TGEV,were constructed.The annealing temperature was optimized,and it was proved that the established system was insensitive to annealing temperature,and the system could be amplified effectively under the four annealing temperature conditions.On the basis of the research method of double RT-PCR detection technology,the quadruple RT-PCR detection method of PEDV,TGEV,PoRV and PDCoV was established by using PEDV,TGEV,PoRV and PDCoV target fragment plasmids as templates,and the gradient of annealing temperature was explored.54℃ was used as the final annealing reaction temperature,and then the primer concentration and enzyme concentration were optimized.after specific verification,the sensitivity test of the quadruple RT-PCR detection method was carried out.The results showed that the detection limits of PEDV,TGEV,PoRV and PDCoV quadruple RT-PCR for the four viral target gene fragments were 7.83× 103、1.82 × 103、8.5× 103 and 8.4×103copies/μL,respectively.The established quadruple RT-PCR method was applied for clinical application.The results were compared with the results of single RT-PCR.The results of PoRV detection were consistent with the results of PoRV single RT-PCR.The coincidence rate was 100%.The detection of PEDV,TGEV and PDCoV was performed.As a result,it was slightly lower than the single RT-PCR results of the three viruses.187 clinical samples were tested and the results showed that the positive rate of PEDV was 8%;the positive rate of TGEV was 3.7%;the positive rate of PoRV was 2.1%;the positive rate of PDCoV was 1.6%.