Simultaneous Typing Of Seven Porcine Virus Pathogens By QIAxcel And Bio-Plex And Its Preliminary Application

In order to detect a variety of porcine viral pathogens including Pseudorabies(PRV),Classical swine fever virus(CSFV),Japanese encephalitis virus(JEV),Porcine reproductive and respiratory syndrome virus(PRRSV),Porcine circovirus type 2(PCV-2),African swine fever virus(ASFV)and Porcine parvovirus(PPV)in one system simultaneously,and promote the capillary gel electrophoresis technique and the liquid chip technique.First of all,seven pairs of specific primers and probes were designed based on the conservative genes including gB of PRV,NS5 of JEV,5’URT of CSFV,ORF1 of PCV-2,ORF6 of PRRSV,VP1 of PPV and P72 of ASFV.Each specific primer was fused to a label sequences at the 5’-end,each probe was amine modified at the 5’-end and included twelve carbon spacers,then a pair of super primer was designed for recognizing the label sequences,and a biotin was substituted at the 5’-end of the reverse super primer.Then the multiple PCR amplification system was preliminarily optimized through routine gel electrophoresis in two groups based on the different length of the amplification fragments.Further,the multiple PCR amplification system was synchronously optimized by QIAxcel capillary gel electrophoresis(PCR-QIAxcel)which is high resolution,and a method for diagnosing and distinguishing PRV,JEV,CFSV,ASFV,PCV-2,PRRSV and PPV simultaneously in one reaction was established.The Bio-Plex suspension array platform based on the multiplex PCR amplification system(PCR-Bio-Plex)was established after hybridization conditions optimizing.Lastly,the sensitivity,specificity,repeatability and clinical application results of these two method were analyzed and compared.The result showed that the best PCR amplification conditions were:final concentration of specific primer and super primer were 0.056 and 0.64 μmol/L/piece,annealing temperature for two stage were 56 and 51 ℃.The best hybridization conditions were 57 ℃ hybridization for 30 min.PRV,JEV,CFSV,ASFV,PCV-2,PRRSV and PPV achieved a sensitivity of 4.56×103,6.35×103,1.98×103,1.32×104,3.21×103,4.51×103,3.33×103copies/μL by PCR-QIAxcel and 2.81×102,2.34×102,2.98×102,2.31×102,2.22×103,2.54×103,2.33×103 copies/μL by PCR-Bio-Plex respectively,both under the optimised PCR and detection conditions.Both of the two method were specific and had no cross-reaction with other viruses.The 65 clinic specimens re-check result showed the positive specimens,which was infectious with at least one of the seven viruses,detected by PCR-QIAxcel reached 47,and the total positive rate was 88.7%(47/53),at the same time,the total positive rate by PCR-Bio-Plex achieved 92.5%(49/53),and the detection consistency of PCR-QIAxcel and PCR-Bio-Plex was 95.9%(47/49).The study established PCR-QIAxcel and PCR-Bio-Plex way for single or mixed detection of PRV,JEV,CFSV,ASFV,PCV-2,PRRSV and PPV simultaneously successfully,then,these two method were evaluated and analyzed.The PCR-QIAxcel and PCR-Bio-Plex are flexible,effective,and relatively specific,sensitive.The sensitivity of PCR-Bio-Plex was one to two orders of magnitude higher than PCR-QIAxcel.This study provide an alternative molecular detection system for these seven pathogens,and provide a new idea and method for the monitoring and diagnose of exotic animal diseases and it has great significance for the development of high-throughput techniques on animal disease diagnosis.