Establishment And Application Of A Multiplex PCR To Determin Porcine Respiratory And Reproductive Disorders

Porcine respiratory and reproductive disorders are the most common and prominent problems in pig production practice in recent years,and have brought enormous harm and loss to China's pig industry.Many studies have shown that swine influenza virus?SIV?,swine fever virus?CSFV?,porcine reproductive and respiratory syndrome virus?PRRSV?,porcine circovirus type 2?PCV2?,porcine parvovirus?PPV?,pseudorabies virus?PRV?,Japanese encephalitis virus?JEV?,etc.cause clinically more respiratory and reproductive disorders in pigs,and often mixed or secondary infections,resulting in high morbidity and mortality of pigs.Make the diagnosis and prevention more difficult.In order to realize the rapid diagnosis of mixed infections of various pathogens of clinical porcine respiratory and reproductive disorders,this study established a seven-plex PCR detection method for the above pathogens and applied it for rapid diagnosis and prevention of related pathogens.Providing effective technical means,and cloning and sequence analysis of some virus-related virulence genes or antigen genes for single or mixed infection positive virus tissue samples detected by seven-plex PCR,and analyzing Guizhou Province from the perspective of molecular biology The genetic variation of some of the porcine respiratory and reproductive disorders viruses that have been popular in recent years provides a theoretical basis for the prevention and control of these viruses in the future.The main research contents are as follows:1.Establishment of seven-fold PCR detection method for PRRSV,PCV2,PPV,PRV,SIV,JEV and CSFVRefer to partial fragments of genes such as PRRSV ORF6?AY262352?,PCV2ORF2?KY806071?,PPV NS1?AY502114?,PRV gB?JX41771?,SIV M?LC371845?,JEV NS1?M55506?and CSFV E2?AF092448?on GenBank,Seven pairs of primers were designed to amplify partial fragments of PRRSV ORF6,PCV2 ORF2,PPV NS1,PRV gB,SIV M,JEV NS1 and CSFV E2 genes.The virus stored in the laboratory,such as PRRSV,PCV2,PPV,PRV,SIV,JEV,and CSFV,was inoculated into the monolayer susceptible cells for virus proliferation,and the nucleic acids in the venom were extracted as templates,and 7 were passed.Identification of virus single PCR amplification products,optimization and detection of annealing temperature and sensitivity,and establishment of a rapid identification of 7 common porcine respiratory and reproductive disorders such as PRRSV,PCV2,PPV,PRV,SIV,JEV and CSFV Single PCR detection method for viroids;based on the method,selected the appropriate seven PCR buffer system and enzyme,explored the annealing temperature,template and primer concentration,and finally tested the specificity,sensitivity and reproducibility of the seven-plex PCR.A multiplex PCR assay capable of simultaneously and rapidly identifying seven common porcine respiratory and reproductive disorders viruses such as PRRSV,PCV2,PPV,PRV,SIV,JEV,and CSFV.The establishment of single virus PCR detection method showed that:?1?the optimal reaction system:7 pairs of primers were 10?mol L·L^-1,PRRSV,PCV2,PPV upstream and downstream primers each 1?L,PRV,SIV,JEV,CSFV The upstream and downstream primers each contained 1.5?L,the template DNA/cDNA was 2?L each,and the gold MiX?Green?supplemented 25?L.?2?Optimum reaction conditions:the specific annealing bands of single virus PCR can be amplified at 50-60°C.The optimal reaction procedures for the seven viruses are:94?5 min;94?45 s;54?45 s;72?45 s,35 cycles;72?10 min?3?Sensitivity results showed that the minimum nucleic acid detection amounts of the seven viruses were:PRRSV 0.47 pg,PCV2 0.33 pg,PPV34 pg,PRV 1.2pg,SIV 0.18 pg,JEV 8.5 pg,CSFV 0.8 pg.The results of the seven-plex PCR assay showed that:?1?the optimal reaction system:the optimal PCR reaction system was 50?L,the upstream and downstream primer concentrations were all 10?mol L·L^-1,and the primers were PRRSV,PCV2,PPV,SIV.Each 0.5?L,1?L of PRV,JEV,and CSFV,DNA/cDNA template:1?L for each of PRRSV,PCV2,PPV,and SIV,1.5?L for each of PRV,JEV,and CSFV,and 50?L for Gold MiX?Green?.?2?Optimum reaction conditions:94?for 5 min;94?for45 s;54?for 45 s;72?for 45 s,35 cycles;72?for 10 min.?3?Specificity,sensitivity and reproducibility test results showed that seven-plex PCR can amplify specific fragments of PRRSV,PCV2,PPV,PRV,SIV,JEV and CSFV,which are 224bp,353 bp,and 424 bp,respectively.549 bp,684 bp,831 bp,1 121 bp,DNA/RNA of E.coli,APP,PEDV,and normal cell tissues were not amplified;the minimum nucleic acid detection amount of each virus in the seven-plex PCR was:PRRSV 94 pg PCV266 pg,PPV 68 pg,PRV 20 pg,SIV 35 pg,JEV 17 pg and CSFV 14 pg;6 replicates were able to amplify a consistent target band.The seven-plex PCR detection method for porcine respiratory and reproductive disorders viruses has the characteristics of high sensitivity,specificity,and reproducibility.2.Application of PRRSV,PCV2,PPV,PRV,SIV,JEV and CSFV Seven-plex PCR Detection Methods in Clinical SamplesSeven-plex PCR methods for the detection of porcine respiratory and reproductive disorders were used to detect 150 clinical samples from parts of Guizhou province from2017 to 2019.The results showed that the positive rate of mixed infection of 150 kinds of clinical diseases was as high as 66%?99/150?,and the positive rate of mixed infection of PCV2+PRRSV was 42.67%?66/150?,mixed infection of PCV2+PRV.The positive rate was 8%?12/150?,the positive rate of PCV2+CSFV mixed infection was2.67%?4/150?,and the positive rate of PCV2+JEV mixed infection was 3.33%?5/150?,PCV2+PPV mixed.The positive rate of infection was 0.67%?1/150?,the positive rate of mixed infection of PRV+CSFV was 2%?3/150?,and the positive rate of mixed infection of PRV+JEV was 5.33%?8/150?;3 viruses The positive rate of mixed infection was 11.33%?17/150?,and the positive rate of mixed infection of PCV2+SIV+JEV was 2%?3/150?,and the positive rate of mixed infection of PCV2+PRV+JEV was 3.33%?5/150?,the positive rate of PCV2+PRRSV+CSFV mixed infection was 1.33%?2/150?,the positive rate of PCV2+PRRSV+JEV mixed infection was 4.67%?7/150?;4 viruses PCV2+PRRSV+PRV+The positive rate of CSFV mixed infection was 2%?3/150?,and the positive rate of mixed infection of 5 viruses PCV2+PRRSV+JEV+PRV+CSFV was 0.67%?1/150?;both the positive rate of mixed infection of 6 and 7 viruses are 0.The coincidence rate of multiplex PCR and single PCR of positive PRRSV,PCV2,PPV,JEV,and SIV was 100%,and the coincidence rate of positive CSFV and PRV was over 90%.3.Study on genetic variation of some epidemic strains of the respiratory tract and reproductive disorders in large-scale pig farmsRefer to the gene sequences of genes such as PRRSV ORF5 and Nsp2?EF635006?,JEV E?M55506?,CSFV E2 gene?AF092448?,PRV gE?KM061380?,and PCV2 whole gene?KU041852?on GenBank,and use primer 5.0 to design for amplification.The primers of the target gene fragment were based on the detection and analysis of mixed infections of porcine respiratory and reproductive disorders,and the positive clinical results of PRRSV,PCV2,PRV,JEV,CSFV single or mixed infection were selected in this study.The disease was used as a template to clone and genetically analyze PRRSV ORF5 and Nsp2 genes,PCV2 gene,PRVgE gene,JEV E gene,and CSFV E2 gene.The amino acid sequences of the four PRV gE genes cloned had an insertion of aspartic acid?D?at positions 48 and 496,and the amino acid sequence of the gE gene of the PRV variant isolated from different provinces since 2012 was 448.The positions are all from V?I and belong to the PRV mutant strain.The eight PCV2 whole gene sequences cloned belong to the PCV2d genotype.The 5 PRRSV ORF5 genes cloned in the protein epitope:2 non-neutralizing epitopes?aa27^aa30 and aa180^aa197?and 1 neutralizing epitope?aa 37^aa 45?no mutations.In addition,the five PRRSV Nsp2 gene proteins have two contiguous amino acid deletion sites at positions 481 and 532 to 560,which are consistent with the deletion sites of the highly pathogenic porcine blue ear virus strain sequence.The same branch,the kinship is also recent.The viral antigenicity and virulence-related amino acid positions of the four CSFV E2 gene sequences cloned did not change greatly.Some of the CSFV E2 genes were 24 G?E,36 N?D,and 45 K?R,49.At the V?T,the E2 gene of the swine fever virus in Guizhou Province is developing away from the classical strain and the vaccine strain and is in the same genetic evolution branch as the Chinese isolates that have been popular in recent years.The cloned four JEV E gene sequences did not mutate in the Cys at both E304,E335 and were responsible for the 10 key amino acid positions of JEV virulence(E107,E138,E176,E177,E244,E264,E279.There was no variation on E315,E439,E447,and the amino acid positions of E60-68 were identical to those of vaccine strain SA14-14-2.