Preliminary Study On Multiple PCR Detection Technology For 7 Pathogens Of Porcine Diseases

Porcine pseudorabies virus,(PRV),Classical swine fever virus,(CSFV),African swine fever virus,(ASFV),porcine reproductive and respiratory syndrome virus,(PRRSV),Haemophilus parasuis,(HPS),Pasteurella multocida,(PM),Actinobacillus pleuropneumoniae,(APP)represent several kinds of major infectious disease in swine industry,caused significant impact on pig industry and induced great economic losses.In order to achieve the goal of rapid,sensitive and accurate detection of these 7 pathogens,two methods consisted of normal multiplex PCR,(mPCR)and capillary electrophoresis multiplex PCR,(mPCR-CE)were used for the preliminary study of detection technology.(1)Development of multiplex PCR for detection of 7 pathogens.Firstly,seven pairs of specific primers were designed through analyzing of the conservative sequences,PRV gB,CSFV E2,ASFV P72,PRRSV M,HPS 16S rRNA,PM PLPE and APP OMLA,respectively.Subsequently,a normal mPCR method was established based on the optimization of annealing temperature,reaction condition and primer concentrations.Use gradient PCR amplification in 50-58 ? to optimize annealing temperature.The sensitivity test was performed by using serially diluted standards(10-1 to 10-8).Specificity test was performed through applying some other common pathogens,including JEV,PPV,PEDV,TGEV,E.coli,Salmonella and Staphylococcus aureus.Cross reactivity test was performed by using random mixed templates that orginate from cDNA or DNA of these 7 pathogens.To assess the consistency of this method,clinical test was carried out by detection of 67 clinical specimens through this mPCR,with a parallel test performed by technology in national standard.Results indicated that the optimum annealing temperature was 51 ?.In sensitivity test,the detection limit was 103-104 copies/?L when all of the seven target bands could be detected simultaneously.The specificity test was carried out by validation of this method through amplification of negative control samples,and all of the detection results of these samples negative.And there were no cross reactivity between the seven pathogens and the seven pairs of specific primers.In the test of clinical samples,67 clinical samples which constituted of 44 multi-infected samples and 14 single-infected samples and 5 negative samples,were detected by this method.The positive rate and consistency were 86.6%and 94%,respectively.(2)Development of capillary electrophoresis multiplex PCR.Firstly the conservative sequences of the 7 reference pathogens were analyzed to design 7 pairs of specific primers.Subsequently,the Specific chimeric primers were obtained by adding universal primer sequences in 5' end of each specific primer.Use gradient PCR amplification in 50-60 ? to optimize annealing temperature.The primer concentrations were optimized by orthogonal test.The sensitivity test was performed by using serially diluted standards(107 to 10-1 copies/?L).Cross reactivity test was performed by using random mixed templates that orginate from cDNA or DNA of these 7 pathogens.Specificity test was performed by detection of some other common pathogens,including PEDV,TGEV,JEV,E.coli,and Staphylococcus aureus.Repeatability test was performed by using serially diluted standards(106-104 copies/?L)as templates to be amplified triplicates,by which the relative fluorescence intensity,concentrations of PCR products and product lengths(bp)were obtained for the calculation of relative variable coefficient.The results indicated that the optimum annealing temperature was 58.3 ?.Detection limit of this assay was 103 copies/?L,when 7 positive results were visible from identical lane.When using a variety of control samples,results of positive samples showed specificity peak in contrast of the negative results of all the negative control samples.In addition,neither cross reactivity nor unspecific amplification were detected between these 7 kinds of random mixed templates.The variable coefficient of relative fluorescence intensity,concentration of PCR products and product size(bp)were 2.93%,3.69%and 0.11%,respectively,which demonstrated that the differences among triplicate experiments were small and lower than 5%.The testing results of the 67 clinical samples were exactly the same with the results that obtained by national standards,with 44 single-infected samples,18 multi-infected samples and 5 negative samples,indicating the positive rate of 92.5%and the consistency of 100%.As is indicated in the results,these two kinds of multiple PCR methods that established in this study can rapidly and simultaneously detect 7 kinds of major swine disease pathogens,showing great specificity,sensitivity and veracity,which provides rapid,sensitive,specific and reliable scientific detection tools and technical reserves for clinical diagnosis,domestic and overseas animal quarantine.