Establishment And Preli Minary Application Of Double Fluorescent Quantitative PCR Detection Method For Torque Teno Sus Virus 2 And Porcine Circovirus Type 2

In order to promote the transformation of animal husbandry production,the standard production of technology for pigs is more and more reasonable.However,the pig breeding environment can not be improved in time,pig infectious diseases is harm to human health.The diagnosis,prevention,and treatment are more and more difficulty,and the traditional diagnostic methods are insufficient.Diagnosis of newly discovered and multiple pathogen infections is particularly urgent.Postweaning Multisystemic Wasting Syndrome?PMWS?is a chronic,progressive,high-fatality disease which is prevalent in pig farms in China in recent years.Its etiology is more complicated,it mainly is infected with porcine circovirus.with other pathogens are also the cause of the disease.Since the discovery of PMWS,research of PMWS pathogens has co ming out.Porcine circovirus?PCV?is highly correlated with PMWS.A large number of PCV2 antigens were found in the pathological tissues of PMWS disease pigs.PCV2 virus was isolated by cell culture,but other pathogens were not isolated.In the case of PMWS,it was found that the clinical manifestations of specific lesions were consistent with the severity of PCV2.The results of PCV2 were frequently detected in PMWS cases and fewer pathogens were detected,indicating that PCV2 is the main cause of PMWS,and infection alone can cause PMWS.Transfusion Transmitted Virus?Torque to the virus,TTV?belong to anelloviridae,anellovirus.It is a non-enveloped DNA virus with a single-stranded,closed loop in the genome.Therefore,the infection of the virus does not immediately cause diseases.However,TTSuVs can influence the development of some diseases and even influence their results.It has not been reported that TTSuVs is directly related to the occurrence of certain diseases in pigs.Because PCV2 can cause the multi-systemic wasting syndrome in weaned piglets,the genetic relationship of PCV2 is adjacent to TTSuVs.PMWS and other related diseases caused by porcine circovirus?PCVD?may be caused by co-infection of PCV2 and TTSuV.In order to establish a dual fluorescence quantitative PCR detection method for PCV2 and TTSuV2 and its preli minary application,As follows1 Establishment of PCV2 real-time quantitative PCR assayThe viral genome was extracted from the diseased animals,and specific amplification primer was designed according to the conserved region of the PCV2sequence published by GenBank,and PCR amplification was performed according to special program.The PCR product was purified and ligated to cloning vector for construction of a standard plasmid.The standard plasmid was used as a template to optimize the primer concentration,annealing temperature and Taq enzyme dosage.The standard curve of real-time PCR was drawn and the real-time PCR detection method of PCV2 was established.optimum system:Premix Ex Taq?2X?:12.5?L,primer:1.0?M,probe:0.75?M,DNA template:3?L,add ddH₂O up to 20 mL.Optimal condition:95?,30 S.95?,10 S.56.6?,30 S,35 cycle.2 Establishment of TTSuV2 real-time quantitative PCR assayThe viral genome was extracted from the diseased animals,and specific amplification primer was designed according to the conserved region of the TTSuV2sequence published by GenBank,and PCR amplification was performed.The PCR product was purified and ligated to a cloning vector for construction of a standard plasmid.The standard plasmid was used as a template to optimize the primer concentration,annealing temperature and Taq enzyme dosage.The standard curve of real-time fluorescent quantitative PCR was drawn,and the real-time quantitative PCR detection method of porcine circovirus type 2 was established.optimum system:Premix Ex Taq?2X?:12.5?L,primer:1.0?M,probe:0.75?M,DNA template:3?L,add ddH₂O up to 20mL.Optimal condition:95?,30 S.95?,10 S.56.6?,30S,35 cycle.3 Preli minary application of double fluorescence quantitative PCR detection method for PCV2 and TTSuV2The DNASTAR software analysis confirmed that the two probes,the four primers did not form a primer dimer with each other,the best annealing temperature,primer and probe concentration were found.Diseases DNA was detected according to established methods in the experiments.the results that PCV2 and TTSuV2 optimal condition:PCV2 primer:1.0?M,TTSuV2 primer:1.0?M.Optimal condition:95?,30 S.95?,10S.55.8?,30 S,35 cycle.Under these conditions,the minimum copy number of double fluorescence quantitative polymerase chain reaction?DFQPCR?was 1X10² copies/?L for the two standard samples.There was no non-specific amplification curve and ross-reaction.The double fluorescent quantitative PCR method was applied to detect 300 serum samples in the pig breeding center and the center for disease control and prevention of jilin Agriculture Science and Technology College in 2017-2018.8 years.The results showed that the infectious rate of PCV2was 36%,the infectious rate of TTSuV2 was 60%,and mixed infectious rate of of two viruses was 14.8%.Two sets of primers,PCV2 and TTSuV2,have good specificity,Clinical trials have shown that this method have better sensitivity and specificity for detection of clinical samples.