Detection And Genetic Analysis Of Classical Swine Fever Virus And Porcine Circovirus Type 2 In Slaughtering Pigs In Shaanxi Province From 2018 To 2019

Classical swine fever（CSF）and Porcine circovirus diseases（PCVDs）are important infectious diseases affecting the pig industry.The pathogens are Classical swine fever virus（CSFV）and Porcine circovirus type 2（PCV2）.There are many changes in the clinical manifestations of CSFV and PCV2 infected pigs under vaccine immunization pressure.Some infected pigs do not show clinical symptoms and become an important source of infection.In addition,virus variation may also be important reason for revaccination of vaccine immunized pigs.Establish sensitive and specific detection methods,which can quickly detect CSFV and PCV2 poisoned pigs and treat them in time.Loop-mediated isothermal amplification（LAMP）is a novel nucleic acid detection technology that is sensitive,specific,and easy to operate for viral nucleic acid detection.It has been widely used in virus detection.Clinically healthy slaughter pigs may also carry CSFV and PCV2.By detecting the carrying rate of the two pathogens and analyzing the genetic variation of the detected virus,it can provide basic information for the prevention and control of CSFV and PCV2 in the farm.This study first established a LAMP method for detecting CSFV and PCV2,and collected samples from large-scale pig slaughter enterprises in Shaanxi Province for nucleic acid detection of CSFV and PCV2.Viral gene amplification and sequence analysis,and isolation and identification of viruses were performed on samples that were positive.The study obtained the following results:（1）An RT-LAMP method for detecting swine fever virus and a LAMP method for porcine circovirus type 2 were established.The established method has a high sensitivity.The minimum detection limit of viral RNA for RT-LAMP method is 2.58×10^-7 ng/μL,and the minimum detection limit for porcine circovirus type 2 LAMP method for viral DNA is 9.7×10^-5 ng/μL;The minimum detection limits of the PCR method were 2.58×10^-6 ng/μL and 9.7×10^-4 ng/μL,respectively.（2）The pig prion and porcine circovirus type 2 were slaughtered in Shaanxi Province from2018 to 2019.In 2018-2019,300 samples of slaughtered pigs from 7 regions were collected from Shaanxi Province and tested by established CSFV and PCV2 LAMP methods.The results showed that there were 30 CSFV positive samples with a positive rate of 10.0%（30/300）and43 PCV2 positive samples with a positive rate of 14.3%（43/300）.The results of routine RT-PCR（PCR）were 21 CSFV positive samples with a positive rate of 7.0%（21/300）and 38PCV2 positive samples with a positive rate of 12.7%（38/300）.（3）Genetic analysis and typing of swine fever virus and porcine circovirus type 2 were performed.The full-length E2 gene fragment was obtained from 12 samples of 30 CSFV positive samples.The gene analysis showed that 2 strains of CSFV belonged to the 1.1 strain of Shimen virulent strain,and 9 strains belonged to 1.1 subtype of swine fever rabbit attenuated vaccine strain.It indicates that the current CSFV in the pigs in Shaanxi Province is mainly 1.1 subtypes.The full-length fragment of PCV2 Cap gene was obtained from 20 samples of 43 PCV2positive samples.Genetic analysis showed that 11 strains belonged to PCV2b subtype and 9strains belonged to PCV2d subtype,indicating that PCV2 in Shaanxi pig population is 2b and2d subtypes are dominant.（4）A porcine circovirus type 2 isolate was obtained.Two PCV2 isolates were isolated from 43 PCV2 nucleic acid positive samples,all of which were PCV2b subtypes,named PCV2/SX-BJ/2019 and PCV2/SX-YL/2019,respectively,which were isolated from infected PK-15 cells.The titers in the range were 10^-5.5994TCID₅₀/100μL and 10^-4.7096TCID₅₀/100μL,respectively.