Establishment Of Multiplex PCR And Gene Chip For Detection Of PCV-2 CSFV And PRRSV

Multiple infections have become widespread in swinery, and have made the conditions more complex. This case increased the difficulty of clinical and laboratory diagnosis. PCV-2, CSFV and PRRSV are primary factors threatening food safety and human health.Therefore, it's necessary to establish a pluralistic method to diagnosis the complex infective diseases. In this study, multiplex PCR and the diagnostic DNA (?)ine viral disease has been constructed.A genome comparison method was used to identify specific PCR target sequences and create detection database of Virus genome. Primers and probes for the detection were selected and validated.Then multiplex PCR and gene chip methods based on selected primers and probes were developed and optimized to detect three mentioned Viral pathogens.Major study reads as follows:Part 1:Multiplication and identification of virusPRRSV,PCV-2,CSFV were multiplicated by using MARC-145,PK-15,ST cells. The viral multiplication were observed by CPE and indirect immunofluorescence technique. The results show that the virus were highly multiplicated.Part 2:Multiplex PCRThe establishment of the multiplex PCR detection method was very important for developing porcine reproductive Problems detection kit. Based on the similarity of the viral sequences deposited in the GenBank database,four pairs of primers were designed. By using four pairs of virus specific primers, four PCR/RT—PCR assay were established to amplify the conservative regions of the four viruses respectively. Consequently.a multiplex PCR/RT PCR method to detect the four viral strains was developed. The sensitivity achieved was as low as 10-4-10-5 and the total detection time was less than 4 hours.Then the 60 clinical samples were identified with the multiplex PCR and single PCR, respectively.The sensitivities of the multiplex PCR and single PCR were compared. The results indicated that the optimal conditions of the multiplex PCR layed a good foundation of sensitive, special and rapid assay for detection of clinical samples. It could be widely applied to clinical inspection.Part 3:Gene chipThe establishment of the Oligonucleotide microarray diagnostic technique was very important for developing porcine reproductive Problems detection kit. Based on the similarity of the viral sequences deposited in the GenBank database, Species-specific 60-mer oligonucleotide probes (oligoprobes) acted as the capture probes were designed and immobilized on the surface of amino modified slides. Primers were designed and labeled with Cy3.Multiplex PCR was used to prepare abundant fluorescently-tagged target DNA.Following purification,the labeled PCR products were hybridized to the microarray. The presence of the virulence genes was determined by a fluorescent signal above the background noise.When the spots of capturing probes was corresponded with samples a positive result displayed,while in the negative and blank controls showed no signal. Then the 60 clinical samples were identified with Oligonucleotide microarray diagnostic technique and PCR/RT-PCR, respectively.The sensitivities of the Oligonucleotide microarray diagnostic technique and PCR were compared (?)liance rate of both was 92%.The results indicated that the Oligonucleotide microarray diagnostic technique could be applied to detection of clinical pathologic samples for diagnosis of PRRSV,CSFV and PCV-2.