Porcine Circovirus Type ?,swine Fever And Porcine Reproductive And Respiratory Syndrome Virus Triple The Establishment Of RT-qPCR Detection Method And Preliminary Application

Immune suppression is a manifestation of the abnormal immune function of livestock and poultry.It will damage the immune function of the body and make the body lack of specific immunity,leading to the failure of vaccine immunization in the farm or the increase in the incidence of infection,causing huge losses to the pig breeding industry.Porcine circovirus type ?(PCV-?),Classical swine fever virus(CSFV)and Porcine reproductive and respiratory syndrome virus(PRRSV)are common immunsuppressive viruses.Clinically,these three viruses are very prone to mixed infection or secondary infection,and they are also very easy to have concurrent and secondary infections with other pathogens.It is difficult to make accurate judgments only by relying on clinical changes and conventional pathogenic detection methods,especially when mixed infections occur,and it is difficult to carry out early and rapid detection,and prone to misjudgment.This article aims to establish a precise and efficient detection method that can detect multiple pathogens in a single tube at the same time,so as to have important significance for the diagnosis and prevention of such diseases.In this study,the complete genome sequences of PCV-?,CSFV and PRRSV from Gen Bank,and designed primers and probes for constructing a fluorescent quantitative PCR system for the conserved regions of the virus.The PCV-?,CSFV and PRRSV genomes were extracted from disease materials,and PCR amplification was carried out with specific primers.After identification,the amplified products were used to construct standard recombinant plasmids.Using recombinant plasmid standard as the template,the single-plex fluorescence quantitative PCR system for PCV-?,CSFV and PRRSV was constructed and optimized.The constructed single-fluorescence quantitative PCR system was used for specific detection,sensitivity detection,repeatability test and standard curve production of single-fluorescence system.On the basis of completing the construction and optimization of single-plex real-time fluorescent quantitative PCR of three viruses,a triple fluorescent quantitative PCR system was established,so that these three viruses could be detected simultaneously in a single tube.The specificity,sensitivity,repeatability and standard curve of the triple fluorescence quantitative PCR system were detected.Finally,the constructed triple fluorescent quantitative PCR system and ordinary PCR system were used for clinical testing at the same time,and the test results of the two system were compared.In this study,the single-plex fluorescence quantitative PCR system constructed for PCV-?,CSFV and PRRSV respectively showed that: 1.The three viruses had no non-specific amplification in the single-plex fluorescence quantitative PCR system.2.The sensitivity of the three viruses in the single-plex fluorescent quantitative PCR system could reach 10 copies/?l;3.The intra-group and inter-group repeatability coefficient of variation of the single fluorescent quantitative PCR system was within 5%;4.Single fluorescence quantitative PCR showed a good linear relationship within the dilution range.The triple fluorescence quantitative PCR system constructed for PCV-?,CSFV and PRRSV showed that: 1.The three viruses had no non-specific amplification in the triple fluorescence quantitative PCR system.2.The sensitivity of the three viruses in the triple fluorescence quantitative PCR system could reach 10 copies/?l;3.The coefficient of variation of repeatability within and between groups was within 5%.4.Triple fluorescence quantitative PCR showed a good linear relationship within the dilution range.The triple fluorescence quantitative PCR system constructed in this study was used for clinical detection of 106 tissue samples,in which the positive detection rate of PCV-? was 74.5%,the positive detection rate of CSFV was 34.9%,and the positive detection rate of PRRSV was18.9%.Among them,the positive detection rate of PCV-?,CSFV mixed infection was 22.6%;the positive detection rate of PCV-?,PRRSV mixed infection was 17.9%;and the positive detection rate of CSFV and PRRSV mixed infection was 6.6%;PCV-?,CSFV and PRRSV The positive detection rate of the three mixed infections was 6.6%.In the ordinary PCR test,The positive detection rates of PCV-II,CSFV and PRRSV were 37.7 %,16.9 % and 7.5 %.The positive detection rate of PCV-? and CSFV mixed infection was 7.5%;the positive detection rate of PCV-? and PRRSV mixed infection was 3.8%;the positive detection rate of CSFV and PRRSV mixed infection was 1.9%;the positive detection rate of PCV-?,CSFV and PRRSV mixed infection was 1.9%.The research results showed that the PCV-?,CSFV and PRRSV triple fluorescence quantitative PCR method established in this study has good specificity,sensitivity and repeatability.And the PCV-?,PRRSV and CSFV triple fluorescence quantitative PCR detection method established in this study was more sensitive and specific than ordinary PCR method,and had better clinical practicability,which could be used for the detection of clinical samples.