Screening Of Aptamers Specifically Binding To PEDV And Its Preliminary Application

Porcine Epidemic Diarrhea(PED)with main characteristics of diarrhea,vomiting and dehydration is an acute and contact intestinal infectious disease caused by the porcine epidemic diarrhea virus(PEDV).Pigs of all ages can be infected,but the neonates younger than 7 days old suffer the most with morbidity and mortality from 90%to 100%.Nucleic acid aptamers(aptamers for short)are single-stranded DNA or RNA fragments that can specifically bind to target substances,screened by the systematic evolution of ligands by exponential enrichments(SELEX)technology.Compared with antibodies,it has the advantages of wide range of binding target substances,low immunogenicity,fast and simple preparation and easy labeling.Therefore,nucleic acid aptamers have wide application respects in the fields of biomolecular detection,disease diagnosis and treatment and drug residue detection.This study aims to screen and prepare PEDV specific nucleic acid aptamers,and provide important technical support for the further research on PEDV antigen detection technology.First,the initial library of aptamers was synthesized.The total length of the aptamers in the library was 76 nt,including the random sequence(30 nt)in the middle,and complementary sequences of the fixed primers were at both ends.The ELISA plate was coated with purified PEDV,the initial ssDNA oligonucleotide library of 10 μM was added.After incubation and elution,the aptamers binding PEDV were obtained,and then the ssDNA aptamers were amplified by symmetric and asymmetric PCR.After 12 rounds of continuous screening,a secondary library of nucleic acid aptamers specifically combined with PEDV was obtained.And then,the double-strand DNA was amplified from the secondary library of aptamers by symmetric PCR,and then was cloned and sequenced.Furthermore,DNAMAN was employed for the sequence analysis and the secondary structure prediction of the obtained aptamers.The results showed that the secondary structure of aptamers with a high repetition rate was mainly small stem-loop structure.Moreover,the ELISA plate was further coated with purified PEDV,and the affinity,specificity and KD value of the aptamers were analyzed by ELISA.Two aptamers with high specificity and strong affinity were obtained.Immunofluoresence assay(IFA)showed that the selected aptamers could specifically bind to intracellular PEDV-N protein.In addition,the two aptamers did not display inhibitory activity on PEDV strains QY2016 and CV777.Subsequently,the results of IFA based on AP2-8 showed that the virus antigens could be detected at 8 h after cells were infected with PEDV,but the fluorescence signal was the strongest at 24～36 h after infection.Sanwich ELISA results based on the aptamer displayed that the detection limits of aptamer for PEDV strains QY and CV777 were 16 TCID50 and 50.6 TCID50,respectively.The results indicate that the aptamers specifically recognized PEDV were obtainted using the microplate-SELEX technology,which provides important technical support and experimental materials for further research on PEDV antigen detection technology.