Development And Application Of Visual Microarray For Simultaneouly Detecting Five Swine Reproductive Failure Diseases

Classical Swine Fever Virus(CSFV),Porcine Reproductive and Respiratory Syndrome virus(PRRSV),Japanese Encephalitis Virus(JEV),Porcine Circovirus Serotype 2(PCV2)and Pseudorabies Virus(PRV)are the most common agents that cause reproductive failure in swines.They can induce some similar clinical signs.Clinically,it is difficult to distinguish those diseases.Therefore,it is essential to develop a more efficient method for diagnosis of those reproductive failure diseases.CSFV,HP-PRRSV,JEV,PCV and PRV were used as research objects in this research,a naked-eye readable oligonucleotide microarrays was developed combined with gold label silver stain(GLSS).A highly standardized microarray has been established by optimizing the preparation and detection procedure.On the basis of this research,we evaluate the quality and preliminary clinical application of the visual microarray.The main content of the research is as follows:1.Design of target gene primers and oligonucleotide probesThe specific primers were designed according to the EO conserved sequences of CSFV,Nsp2 conserved sequences of HP-PRRSV,PrM/M conserved sequences of JEV,ORF2 conserved sequences of PCV,gB conserved sequences of PRV.The 45bp virus-specific oligonucleotide probes were designed on the basis of unique sequences internal to the amplified regions,fifteen T bases were inserted into the probes,as well as the 5 ’ end of each probe was modified with an amino group.2.Construction and optimization of visual microarray(1)Asymmetric PCR amplification techniquesA large number of single-stranded target genes biotin-labeled could be prepared by asymmetrical PCR by optimizing the concentration of forward primer and reverse primer.The production of single-strand target genes biotin-labeled reached its peak of at proportion of 1:8 for CSFV-EO,while JEV-PrM/M、HP-PRRSV-Nsp2、PCV-ORF2 and PRV-gB at proportion of 1:6.(2)Preparation of visual microarrayThe oligonucleotide probes,was diluted to a final concentration of 50μmol/L with Baio(?)spotting buffer and ddH2O.The oligonucleotide probe was spotted onto an aldehyde substrate by SmartArrayerTM according to the pre-designed pattern.The aldehyde-modified glass slides were incubated in a humid chamber at 37℃ overnight after UV cross-linking.The aldehyde groups was sealed by 0.5%NaBH4 and kept at 37C for 30min.The prepared microarrays were stored at 4℃ after washing process and drying.(3)Optimization of visual microarray reaction conditionsA standardized visual microarray detection method was developed,with the condition optimized.The 50μmol/L oligonucleotide probes were immobilized on the surface of the aldehyde-modified glass slides to prepare microarray.The microarray was hybridized by target gene for 120min at 45℃;Streptavidin-nangold at a concentration of 4μg/mL that diluted 20 times was incubated at 37℃ for 30min.Under the condition of dark,silver stain reacted for 6-8min at 37℃.3.Evaluation of visual microarrayIn this study,the specificity,sensitivity and shelf-life time of visual microarray was evaluated.The microarray was evaluated by detecting the clinical samples and compared with the results detected by PCR/RT-PCR.(1)Quality evaluation of visual microarrayThe CSFV,HP-PRRSV,LP-PRRSV,JEV;PCV2,PRV and PPV were used as templates for PCR amplification.Then,the labeled products were hybridized with microarray to evaluate the specificity;The recombinant plasmids were diluted with ddH2O to a different concentration,and then labeled with asymmetric PCR.After hybridization with the microarray,the sensitivity was evaluated.The microarrays prepared in the same batch were restored for 15 days,30 days and 45 days,60 days,the labeled productions were hybridized with microarray to detect the shelf-life time for microarray.The results indicated that there were only black silver staining signals at the corresponding positions of CSFV,HP-PRRSV,JEV,PCV2 and PRV,and the results of LP-PRRSV and PPV were negative,and there was no interference among the oligonucleotide probes.The sentivity for visual microarray was 25.0pg/μl.The microarray can be effectively detected after 60 days of storage.(2)The Preliminary clinical application of visual microarrayIn this study,113 clinical samples were collected from Mianyang,Pixian,Yibin,Qionglai,Xinjin,Mingshan,Beichuan,Chongzhou,Tongnan,Hongya and Anyue.Genomic RNA and DNA were extracted from liver,lung,kidney,spleen and other tissues of dead pigs.RNA virus was reverse transcribed into cDNA,then cDNA/DNA was used as templates for asymmetric PCR amplification.Then,the labeled products were hybridized with microarray to compare the coincidence with PCR/RT-PCR.The results indicated that the specificity of visual microarray was 100%and the sensitivity was over 84%.The total coincidence rate of PCR/RT-PCR and microarray was higher than 96.5%.The results indicated that the visual microarray could be used for detecting clinical samples.