Isolation,Identification Of Porcine Acute Diarrhea Syndrome Coronavirus And Establishment Of The Multiple Fluorescence Quantitative PCR For Detection Of Four Porcine Coronavirus

Coronavirus are a class of single-stranded RNA viruses with envelope,which are divided into four genera:α,β,δandγ.Coronaviruses of different genera have no cross protection.It infects humans,pigs,other mammals,poultry and birds,causing intestinal or respiratory diseases.Among them,the coronavirus that can infect pigs has a wide distribution range and high mortality,which seriously threatens the production safety of animal husbandry.Porcine epidemic diarrhea virus（PEDV）,porcine transmissible gastroenteritis virus（TGEV）,porcine respiratory coronavirus（PRCV）,porcine acute diarrhea syndrome coronavirus（SADS-CoV）and porcine enteric coronavirus（Se CoV）belong toαcoronavirus.Porcine hemagglutinating encephalomyelitis virus（PHEV）is aβcoronavirus,and porcine Delta coronavirus（PDCoV）is aδcoronavirus.Most of the above-mentioned coronaviruses are usually clinically manifested as gastrointestinal symptoms in piglets.Since these kinds of porcine coronaviruses are usually indistinguishable in terms of virus morphology,and the clinical symptoms caused by a single coronavirus or a combination of several coronaviruses are very similar,it is impossible to make an effective differential diagnosis of infected animals only by clinical symptoms and virus morphology.Therefore,it has important clinical application value for accurate and rapid differential diagnosis of different coronaviruses in the accurate diagnosis of porcine viral diarrhea.Objective:To isolate and identify a SADS-CoV virus isolate;to establish a multiplex detection method that can detect multiple porcine coronaviruses simultaneously in a short period of time using fluorescence quantitative PCR detection technology will help promote the standardization of the domestic experimental pig pathogenic microorganism detection system and improve the microbiological quality control standard of experimental pig.Methods:（1）The supernatant from the pathologic specimen of sick pigs with diarrhea symptoms was taken after freezing and thawing.It infected Vero cells in a maintenance solution with a final concentration of 5μg/m L of trypsin treated with TPCK,and the cytopathic conditions were observed periodically.The virus strain was collected by freezing and thawing after most cells appeared cytopathic effect,its virus titer was detected when being identified as positive by RT-PCR.The whole gene of the SADS-CoV strain isolated was sequenced,and the sequencing results were compared with other strains.（2）In this study,the whole genome sequences of epidemic strains of SADS-CoV,PEDV,PDCoV and PHEV recorded in Gen Bank at home and abroad were collected and collated.Biological software was used to design multiple fluorescence quantitative PCR primers and probes.The porcine coronavirus plasmids were constructed for the establishment of detection reaction system,then their homogeneity as standard plasmid was verified by variance analysis.The standard porcine coronavirus plasmid was used as templates for the detection of SADS-CoV,PEDV,PDCoV and PHEV by single fluorescence quantitative PCR,and the amplification efficiency,specificity,sensitivity and repeatability of each single reaction system were tested.（3）Based on the single fluorescence quantitative PCR detection methods,a multi-fluorescence quantitative PCR detection method for simultaneous detection of four kinds of porcine coronavirus was established,and the amplification efficiency,specificity,sensitivity and repeatability of the multi-reaction system were tested.The same samples and primers were detected by PT-PCR,and the performance of the two detection methods was compared according to the experimental results.Result:（1）The SADS-CoV virus was isolated and identified from the intestinal contents of clinical pigs.The virus was proliferated stably on Vero cells and showed obvious cytopathic effect,and its virus titer was 10^3.49TCID₅₀/0.1m L.The whole gene of the SADS-CoV strain was sequenced and the comparison of the sequencing results showed that the SADS-CoV strain was an epidemic strain.Its pathogenicity requires the next step to established a challenge model.（2）Four single fluorescence quantitative PCR methods were established respectively for the detection of SADS-COV,PEDV,PDCoV and PHEV.The correlation coefficient（R²）of the standard curve equation established in the single systems were more than 0.99,showing good specificity,repeatability and sensitivity.（3）In single fluorescent quantitative PCR detection methods on the basis of the established four porcine coronavirus detection at the same time of multiple fluorescence quantitative PCR detection method,each virus amplification reaction has good amplification efficiency,specificity of the experimental results showed that the specific primers and probes only make target gene sequences appear the amplification curve.Sensitivity results showed that the multiplex fluorescence quantitative PCR method could detect the lowest levels of the four coronaviruses is 10² copies/μL.Repeatability results showed that the coefficient of variation within and between groups was less than 2%.It proved that the established multi-fluorescence quantitative PCR assay had good specificity,sensitivity and repeatability.Compared with the experimental results of RT-PCR detection methods,multiple fluorescence quantitative PCR reaction has better method performance.Conclusion:Four single and one multiple fluorescence quantitative PCR methods were established for detection of SADS-CoV,PEDV,PDCoV and PHEV.The detection method can accurately of the four common coronavirus in pig rapid detection,and multiple fluorescent quantitative PCR system can simultaneously detect and distinguish the single coronavirus or mixed infection situation.It has the stronger application value,for this kind of pathogen infection outbreak early diagnosis and epidemiological investigation is of great significance.