Establishment And Preliminary Application Of The Multiple RT-PCR For Detection Of Four Porcine Coronavirus

Transmissible gastroenteritis virus(TGEV),Porcine epidemic diarrhea virus(PEDV),Porcine hemmagglutinating encephalomyelitis virus(PHEV)and Porcine deltacoronavirus(PDCoV)belong to the Coronaviridae coronaviruses and cause clinical symptoms of pigs with watery diarrhea and vomiting.Currently,there are no effective prevention and control measures,causipg serious economic losses to the pig industry.A growing number of studies have shown that the four viruses are often mixed infections in the clinic,which poses a huge challenge for the differential diagnosis of the disease.At present,most of the detection methods established for the aforementioned viruses are directed against a single virus,and there are fewer joint detection methods.Considering that all four viruses are morphologically indistinguishable from each other and the clinical features of infected piglets have certain similarities.Therefore,this study started with a differential diagnosis method and established a method for simultaneously detecting and distinguishing four types of piglets.The multi-link RT-PCR method for detection of coronaviruses and their preliminary use in clinical sample detection are important for early epidemic diagnosis and epidemiological investigation of such pathogenic infections.The study obtained the following results:Accordipg to the full gene sequences of four coronaviruses(TGEV,PEDV,PHEV and PDCoV)published in GenBank,various biological softwares were used to design and select primers for the conserved regions of the four viruses.The four virus-positive plasmids were constructed and the plasmids were sent to a biotechnology company for sequencing.The sequencing results were correct.By optimizing the primer concentration and annealing temperature in the RT-PCR reaction system,a single RT-PCR detection method for TGEV,PEDV,PHEV and PDCoV was established.The specific result was that the four single-plex RT-PCR methods only amplified the target gene but not other pathogens.Sensitivity results for single RT-PCR of TGEV,PEDV,PHEV,and PDCoV can detect 3.28pg?0.284pg?25.7pg and 0.592 pg.On the basis of four sipgle-plex RT-PCR reactions,sipgle-stranded development was gradually followed by dual RT-PCR and triple RT-PCR methods.Establishment of dual RT-PCR methods(TGEV-PEDV,TGEV-PHEV,PEDV-PHEV,TGEV-PDCoV,PEDV-PDCoV,and PHEV-PDCoV)and triple RT-PCR methods(TGEV-PEDV-PHEV,TGEV-PEDV-PDCoV,TGEV-PHEV-PDCoV and PEDV-PHEV-PDCoV)and simultaneously optimize the annealing temperature.Explore whether multiple pairs of primers will produce different results due to temperature changes in the same reaction.The results showed that,in dual RT-PCR and triple RT-PCR reactions,templates and primers do not cross-react with each other with different primer pairs and template types at different temperatures,and do not produce dimers with changes in temperature.A multiplex RT-PCR method for the simultaneous detection of the four coronaviruses TGEV,PEDV,PHEV and PDCoV was established based on the above experimental basis.And through the optimization of primer concentration and annealing temperature,the optimal reaction conditions and reaction system were determined.The specific results show that this method can specifically amplify the band of interest.The sensitivity results showed that the multiplex RT-PCR method could detect the lowest levels of TGEV,PEDV,PHEV and PDCoV of the four coronaviruses 32.8 pg?28.4 ng?257 pg and 592 pg.42 clinical samples were tested by single-plex RT-PCR and multiplex RT-PCR using TGEV,PEDV,PHEV,and PDCoV,respectively.From the results,the highest positive rate of PEDV was 42.86%.The positive detection rates of TGEV,PHEV,and PDCoV were 16.67%,16.67%,and 0,respectively.Comparison of the two types of RT-PCR methods showed that thecompliance rate of the four viruses was 100%.