Development And Application Of Visual Microarray For Simultaneouly Detecting PEDV,TGEV And GAR

Porcine epidemic diarrhea virus(PEDV).Transmissible gastroenteritis virus(TGEV)and Group A rotavirus(GAR)is the main pathogen of viral diarrhea piglets.Viral diarrhea has caused a certain degree of loss in the pig industry widely.Because of the similar clinical symptoms of these three piglets’ diarrhea viruses,a rapid detection and identification method of three kinds of viruses is urgently needed.In this research.PEDV,TGEV and GAR was as a research object,a PEDV-TGEV-GAR simultaneous detection visual microarray combined with gold label silver stain(GLST)and asymmetrical PCR was developed.Besides,the preparation and detection methods were optimized,the microarray was used to evaluated by clinical applications.The research main content is as follows:1.The construction and optimization of visual microarray(1)The design of target gene primers and probesOn the basis of previous research,according to the M and S conserved sequences of PEDV,S and N conserved sequences of TGEV,VP7 and NSP4 conserved sequences of GAR recorded in Genbank,specific primers were designed.According to each positive strand of target fragment sequence,two 45 bp length detection probes were designed respectively.(2)The construction and optimization of visual microarray detection technologyIn this research,the standardized construction and detection method of visual microarray was explored and optimized respectively.The standardized construction of visual microarray,the amplification labelling method of single-strand target gene,the standardized detection method and judgement standard was determined.According to the sequence of each target flagment,two 45 bp length oligonucleotide probes were choosen to amino modified with 15 T bases at their 5’-ends and spotted on the designed microarray after mixed with probe solution.Recombinant plasmids DNA was extracted as a template to labelling the target gene with biotin using asymmetrical PCR with designed primers.In asymmetrical PCR system,each reverse primer was labeled with biotin at its 5’-ends.a large number of single-strand target genes with biotin were producted according control the concentration of forwad primer and reverse primer.Observation after hybridization and gold label silver stain.2.The results of construction and optimization of visual microarray detection technologyThe results indicated that the various probes including positive and negative controls were mixed with probe solutions in a 1:1 ratio and then sprayed on the aldehyde-modified glass slides in a designed array.The glass slides with the probes array were incubated in a humid chamber at 37 °C for 10 h,and then deactivated the surface by immersion in a fresh 0.5%solution of NaBH4,25%Absolute Ethanol and 0.75 X PBS buffer.After the optimized wash system,the unbound oligonucleotides probes were removed.The prepared gene chips were stored at 4 °C after 600 r/min centrifugal drying.The production of single-strand biotinylated target DNAs at 1:20 reached its peak of all the dilution rates,except PEDV-M gene(1:10).The certain detection process was hybridized at 45 °C temperature in 90 min,and then completed cleaning solution.Followed by combined with 4 μg · mL-1 nanogold particles modified with streptavidin and stained 12 min-14 min by silver buffer after washed by ddH2O gently.According to the positive control formed black-grey signal spots and negative control did no react,the microarray detection quality and results judgement was detetmined.3.The evaluation of visual microarrayIn this research,the sepecificity,sensitivity and retention period of visual microarray was tested,the microarray was evaluated by clinical samples and compared with the results detected by RT-PCR.(1)Specificity,sensitivity and retention period testSpecificity,sensitivity and shelf of the constructed microarray was tested respectively in this research.Microarrays were hybridized with PEDV,TGEV,GAR,CSFV,PRRSV,PCV2 and JEV products with biotin to test their specificity;each PCR amplification of probe gene plasmid DNA,respectively for 10 x series times diluted then hybridized with chip to detect the sensitivity;the storaged chips of 30 days,60 days,90 days,120 days and 180 days were hybridized for the shelf life testing.The results showed that:Chips hybridized with PEDV,TGEV,GAR were positive and negative with CSFV,PRRSV,PCV2 and JEV.There is no cross-reactivity between each probe;The sensitivity of visual microarray is 22.8 pg/μL;the microarray could still be effectively detected after saved for 180 days.(2)Detection of clinical samplesTo determine the applicability of the detection method,visual DNA microarray was tested to detected 225 clinical samples from 21 regions in Sichuan provinc.Small intestines and intestinal contents were collected from sucking pigs and all samples were assayed with the microarray and reverse-transcription PCR after the RNA was extracted The reverse-transcription PCR results were used to validate the microarray.The result showed that PEDV,TGEV and GAR positive rate was 64.89%,including PEDV infection rate was 60.45%,mixed infection with PEDV and TGEV was 4.4%,no GAR infection.The results showed that PEDV nfection clinically were very common.The coincidence rate of visual microarray and RT-PCR was 100%,which indicated that structured visual microarray could be used in clinical sanmples detection.