Establishment And Preliminary Application Of PRRSV,SS And Pm Multiplex TaqMan Real-Time PCR Detection Methods

Porcine reproductive and respiratory syndrome virus(PRRSV),Streptococcus suis(SS)and Pasteurella multocida(Pm)are three pathogens that are common in the process of pig breeding in China and cause similar clinical manifestations,and clinical differential diagnosis is very difficult.Therefore,it is of great practical value to establish specific,sensitive and rapid detection methods that can diagnose these three pathogens at the same time.This study aims to establish a real-time PCR method that can simultaneously detect PRRSV,SS and Pm pathogens through condition optimization.The details are as follows:1.Establishment of PRRSV,SS and Pm multiplex TaqMan real-time PCR detectionmethodsThrough MEGA7.0 analysis of the gene sequences of three pathogens registered on NCBI,Using the ORF6 gene of PRRSV,GDH gene of SS and KMT1 gene of Pm,three pairs of specific primers and three TaqMan probes were designed and plasmid standards were constructed,and a fast and reliable multiplex TaqMan PCR detection method that can detect PRRSV,SS and Pm was established by optimizing the probe concentration,primer concentration and annealing temperature.The multiplex real-time PCR detection method established in this study had a total reaction system of 20μL,The optimal concentrations of primers specific to PRRSV,SS and PM were 0.4μmol/L,0.3μ/mol and 0.5μmol/L,respectively,and the optimal concentrations of probes were 0.5μmol/L,0.4μmol/L and 0.5μmol/L,respectively,and the amplification effect was best at annealing temperature of 52℃.The correlation coefficients of the standard curves PRRSV,SS,and Pm of the established method are0.999,0.999,and 0.996,showing a good linear relationship The proposed method did not cross-react against various pathogens such as pseudorabies virus porcine circovirus type 2,porcine circovirus type 3,Lawsonia intracellularis,porcine encephalomyelitis virus and Escherichia coli.Repeatable experiments confirmed that the established method had good stability,and the coefficient of variation was less than 1.85%within and between groups.The minimum copy numbers of plasmid standards detected in the effective range(CT≤35)were PRRSV 9.93×10~2copies/μL,SS 1.10×10~2copies/μL and Pm 1.07×10~2copies/μL.2.Preliminary application of PRRSV,SS and Pm multiplex TaqMan real-time PCR detection methodsIn order to verify whether the established method can be applied to clinical testing,multiple TaqMan fluorescence PCR,PRRSV national standard RT-PCR detection method and SS and Pm general PCR methods were used to test 140 clinical samples collected from some pig farms in Guigang City.The detection rates of PRRSV,SS and Pm were40.00%(56/140),30.71%(43/140)and 11.42%(16/140),respectively.The detection rates of ordinary PCR were PRRSV 30.71%(43/140),SS 21.42%(30/140)and Pm 11.42%(16/140).The results show that the detection rate of pathogens by the method established in this study is higher than that of ordinary PCR,and the established method can be used for preliminary clinical detection.On this basis,a total of 1053 clinical samples collected from some pig farms in Guigang City,Guangxi Province were tested using the method established by this institute.The results showed that PRRSV 10.35%(109/1053),SS10.63%(112/1053)and Pm2.09%(22/1053)of the positive samples of simple infection or co-infection were detected,respectively.3.Separation and identification of PRRSV,SS and PmAccording to the above test results,some positive samples were selected and 1PRRSV,20 SS and 5 Pm were isolated.Animal regression experiments showed that SS9and Pm A had different degrees of pathogenicity to BALB/c mice.According to the genetic evolution analysis of ORF5 sequence of PRRSV,the results showed that the isolated strain GG0214 was the closest relative to NADC34,and the nucleotide homology of ORF5 was 93.70%.The results of SS typing showed that among the 20 SS isolates,SS9 accounted for up to 55%(11/20),SS4 and SS12 accounted for 10%(2/20),SS5 and SS27 accounted for 5%(1/20),and 15%(3/20)were untyped.In addition,all 5 isolated Pm strains were type A.Susceptibility experiments showed that 20 SS isolates had the highest resistance rate to tetracycline,erythromycin and lincomycin,all of which were100%(20/20),followed by doxycycline hydrochloride 95%(19/20),florfenicol and penicillin G sodium 40%(8/20)and 15%(3/20),respectively,amoxicillin,ampicillin and enrofloxacin were 5%(1/20),sensitive to ceftiofur sodium and chloramphenicol.The five Pm isolates were 80%(4/5)sensitive to tetracycline,and completely sensitive to penicillin G sodium,amoxicillin,ampicillin,doxycycline hydrochloride and florfenicol.In summary,this study successfully established a real-time PCR detection method for PRRSV,SS and Pm multiplex TaqMan,and applied it to preliminary clinical detection,combined with the results of pathogen isolation and identification,drug resistance and pathogenicity,to preliminarily clarify the infection status of PRRSV,SS and Pm in Guigang.