Simultaneous Typing Of Porcine Reproductive Pathogens By Multiplex PCR And Multiplex PCR With A GeXP Analyser

In order to detect a variety of porcine reproductive pathogens in a system,two detection methods were designed in this study,including simultaneous typing of classical swine fever virus(CSFV),African swine fever virus(ASFV),porcine reproductive and respiratory syndrome virus(PRRSV),highly pathogenic porcine reproductive and respiratory syndrome virus(HP-PRRSV),porcine pseudorabies rabies virus(PRV)by multiplex PCR and simultaneous typing of porcine pseudorabies rabies virus,classical swine fever virus.African swine fever virus,porcine reproductive and respiratory syndrome virus,porcine parvovirus(PPV),porcine circovirus type 2(PCV-2),Japanese encephalitis virus(JEV)by multiplex PCR with a GeXP analyser.Both methods have advantages and disadvantages,and complementary advantages.The application can be selected according to the actual situation,and it can improve the detection efficiency.Firstly,to establish a multiplex PCR for simultaneously typing of five porcine reproductive pathogens,four pairs of primers were used to detect five porcine reproductive pathogens.Specific primers for each of three virus genomes:E2 of classical swine fever virus(CSFV),VP72 of African swine fever virus(ASFV)and gB of pseudorabies(PRV)were used for testing procedure.One pair of primers on NSP2 was designed for two different types of porcine reproductive and respiratory syndrome virus that respectively were porcine reproductive and respiratory syndrome virus(PRRSV),highly pathogenic porcine reproductive and respiratory syndrome virus(HP-PRRSV).After optimizing reaction conditions,a multiplex PCR for simultaneously typing of GSFV,ASFV,PRRSV,HP-PRRSV and PRV was established.The sensitivity of the multiplex PCR was 2.10×103,1.30×103,1.09×104,1.50×103 and 8.97×102 copies/μL for HP-PRRSV,PRRSV,CSFV,ASFV and PRV,respectively.Among 49 clinical samples from Si Chuan provinces co-infection by two or three viruses was 51%(25/49).There were no spurious PCR amplification reactions among all five pathogens noticed with various amounts of DNA and RNA mixtures and all the uninfected controls were scored negative.This method is a rapid,sensitive,specific and cost-effective diagnostic tool for single and mixed infections.And it has great significance for diagnosis and epidemiological investigation of reproductive pathogens in swines.Secondly,to establish a multiplex PCR with a GenomeLab Gene Expression Profiler(GeXP)analyser for simultaneously typing of PRV,CSFV,ASFV,PRRSV,PPV,PCV2,JEV,seven pairs of chimeric primers and one pair of universal primers were designed.Seven pairs of chimeric primers,consisting of a pathogen-specific sequence fused to a universal sequence at the 5’end,were used to initiate the PCR,after which a set of universal primers was used for the subsequent cycles of the PCR.Amplification products were separated by capillary electrophoresis and identified using fluorescence spectrophotometry.The specificity of the GeXP assay was examined with single and mixed pathogen cDNA/DNA templates.The specific DNA product amplification peaks of seven pathogens were observed on the GeXP analyser.Negative controls did not produce DNA products.The sensitivity was evaluated by performing the assay on serial 10-fold dilutions of the plasmids containing the target sequence.Under optimised conditions this assay achieved a sensitivity of 100-1000 copies/μL for a single virus and 1000 copies-μL when all of the seven pre-mixed viral targets were present.Furthermore,the GeXP-PCR assay was 100%specific when 64 clinical samples were tested in comparison with the conventional PCR method.In conclusion,the GeXP assay is a rapid,cost-effective.sensitive,specific and high throughput diagnostic tool for single and mixed infections.