Establishment And Application Of Nano-fluorescence High-throughput Ultrasensitivity Detection Technology For Porcine Major Pathogen

With the development of the large-scale swine industry,porcine viral diseases have shown a gradual and outbreak epidemic.Single infection or mixed infection caused by Classical swine fever virus(CSFV),Porcine reproductive and respiratory syndrome virus(PRRSV),Porcine circovirus type 2(PCV2),Porcine parvovirus(PPV),Pseudorabies virus(PRV),Porcine epidemic diarrhea virus(PEDV)and Transmissible gastroenteritis virus(TGEV)have become the main threat of large-scale swine farm,which bring huge economic losses and severely restricte the healthy development of the swine industry in China.However,existing detection methods can not accurately diagnose pre-clinical samples in the early stage of infection,which would be likely to cause regional outbreaks.Therefore,this study was aimed to establish a specific single or multiple detection and differential diagnosis technology for the above pathogens.To this end,a method was proposed by effectively combining magnetic particle pathogen enrichment technology,nano-gold particle pathogen signal amplification technology and fluorescent probe quantitative PCR technology.It will provide technical support for epidemiological screening and early prevention and control of major swine diseases.First,based on the whole genome sequences of the above seven pathogenic representative strains in the Gen Bank,homology analysis was performed to find highly conserved sequences as design sites for Probes and Oligos.The Probes and Oligos were labeled on magnetic particles(MMPs)and gold nanoparticles(AuNPs),respectively,and then standard strains were used to establish a hybridization reaction system to elute a large number of pathogen-specific Oligos on the AuNPs.And fluorescent quantitative PCR was performed using the designed specific detection MFQ-probe and detection primers to further amplify the pathogen specific signal multiple times,thereby the detection sensitivity was further improved.Hence,the ultrasensitive nanoparticle DNA probe-based fluorescence quantitative PCR assay(UNDP-FQ)designed for preclinical samples was established.Finally,the specificity,sensitivity,reproducibility and clinical application value of the established method are tested by clinical and preclinical samples.On this basis,an ultrasensitive nanoparticle DNA probe-based mixed scale fluorescence quantitative PCR assay(UNDP-MFQ)was further established,which allows for simultaneous detection of CSFV,PRRSV,PCV2,PRV and PPV.The study achieved the following results:1. UNDP-FQ were established for CSFV,PRRSV,PCV2,PRV,PPV,PEDV,and TGEV,respectively.Based on the highly conserved regions of the above seven viruses,14 Probes and Oligos were designed to be labeled with MMPs and AuNPs to form functionalized MMPs and AuNPs.Pathogenic nucleic acids were enriched by functionalized MMPs,pathogenic signals were amplified by functionalized AuNPs,and Oligos were detected by fluorescence quantitative PCR using specific MFQ-probe,which determined 7 optimal Probes and Oligos for UNDP-FQ detection system for the viruses above.The detection limit of UNDP-FQ is 10~2～10~3 times more sensitivity than the currently known Real-time PCR,showing better specificity without cross-reaction with other viruses.The results of repeated tests showed that the maximum coefficient of variation(CV)of within groups and between groups are less than 1%,indicating that the established UNDP-FQ has good repeatability,and the reagents in the system have good stability when stored at 4 ~oC for 6 months.On this basis,for preclinical serum or stool samples,the UNDP-FQ showed a higher diagnostic accuracy and positive detection rate than conventional Real-time PCR.2. Aiming at the five main virusess causing swine reproductive dysfunction disease,theUNDP-MFQ was established to simultaneous detection of CSFV,PRRSV,PCV2,PRV and PPV in the same reaction system at the same time on the basis of the establishment of UNDP-FQ,with the modification of Oligos and the addition of corresponding Taqman Probes further enhanced the specificity of the detection system.First,it was determined the optimal probes and Oligos for viruses above through the UNDP-MFQ detection and screening.The specific detection test showed the UNDP-MFQ only displays the corresponding pathogen specific positive amplification curve without cross-reaction with other pathogens;the sensitivity test showed that the detection limit of UNDP-MFQ is 20copies/m L,which is 5×10~3 times more sensitive than the currently known multiple Real-time PCR;repeatability test showed that the maximum coefficient of variation is less than 1.5%,indicating that the established UNDP-MFQ has good repeatability.For pre-clinical serum samples,the UNDP-MFQ showed a markedly higher diagnostic accuracy and detection rate than conventional multiple Real-time PCR without nucleic acid extraction and reverse transcription.In this study,specific UNDP-FQ were established for seven common pathogens of swine.On this basis,the UNDP-MFQ that can simultaneously detect five viruses of swine reproductive disorders for the early diagnosis and screening needs was established.UNDP-FQ and UNDP-MFQ have the technical advantages of no pathogenic nucleic acid extraction,high sensitivity,strong specificity,and good repeatability,and the reagents have good stability when stored at 4 ~oC for 6 months.It can be used for the diagnosis of preclinical poisoned animals,improve the positive detection rate of preclinical samples,and provide technical support for the early,rapid,high-throughput detection and differential diagnosis of major swine epidemic pathogens,thereby reducing economic losses and providing a set of feasible technologies for preclinical scaled screening of major swine diseases.