Study On Selection And Application Of Aptamers Against Inactive Vibrio Alginolyticus By SELEX

Vibrio alginolyticus is one of the most usual pathogen in aquaculture for its abundant quantity, high pathogenicity and serious infection. Therefore, it is of the upmost importance to detect V. alginolyticus in water and seafood. Based on the characteristics of high affinity and specificity, aptamer has a great potential in the detection of the microoganism. In the present study, aptamers against inactive V. alginolyticus were selected from a 82 nt ssDNA random library by systematic evolution of ligands by exponential enrichment(SELEX). After 15 rounds of selection and amplification, the affinity of aptamers in the last enriched pool increased from 0.025 to 0.452, which increased by 17 times. The last enriched pool was also proved to have good specificity to its target, inactive V. alginolyticus, and its Kd was 27.5 ± 9.2 nM.After cloning and sequencing, fifty-three sequences were obtained from 55 randomly picked clones, but only 32 unique sequences were found. There were six aptamers with a high frequency(#4, #7, #8, #9, #14, #23), which were appeared more than twice. These six sequences(aptamers #4, #7, #8, #9, #14, #23) were proved to have high affinity and specificity with V. alginolyticus, whose dissociation constant(Kd) are 7.14±2.73 nM, 28.39±11.46 nM, 12.82±6.00 nM, 15.21±4.60 nM, 14.65±4.90 nM, 46.89±15.87 nM respectively. Based on the primary structure of these 32 aptamers, they could be divided into nine families. The families from the 1st to the 6th, have shared >80% homology and eight families have conserved sequence. Analysis on the secondary structure of these aptamers showed that the largest number of aptamers was the type P(1+1), which would be amplified more easily.Based on the aptamer enriched pool and PCR technology, qualitative detection of V. alginolyticus was carried out and the results showed that the PCR product of the last selection could detect 102cell/ml inactive V. alginolyticus and 103 cell/ml V. alginolyticus while aptamers could detect 10 cell/ml inactive V. alginolyticus or V. alginolyticus. Aptamers also had a good application in the detection of the pathogen in the serum of fish. Finnly, the qualitative detection technology of V. alginolyticus, which based on PCR technology and aptamers, were established.