| With the rapid development of aquaculture industry,the disease caused by Aquatic Pathogenic bacteria is becoming more and more serious,which brings incalculable economic losses to the aquaculture industry.Vibrio splendidus,as a conditionally pathogenic bacteria,is distributed in salt lakes and oceans,with a wide range of hosts.It can infect bivalves,sea cucumbers,fish and other marine animals,and cause them to die in batches.At present,the detection methods of Vibrio splendidus mainly include the immunology detection technology based on protein level(ELISA,Dot-ELISA,ICA,FA,IFA,etc)and the molecular biological detection technology based on gene level(nucleic acid probe detection technology,PCR,LAMP,etc).However,these technologies have a series of disadvantages,such as high cost,long time-consuming,relying on professionals and testing equipment,and prone to false positive.Therefore,it is urgent to establish a simple,efficient,rapidand safe new detection technology for Vibrio splendidus.In this study,Exo III was used for cyclic enzyme digestion and nano gold labeled DNA probe detection,a sensitive and efficient nucleic acid test strip detection method was established to realize the visual and rapid detection of Vibrio splendidus.The detection signal was amplified by cyclic enzyme digestion reaction,and the visible detection was realized by the gold labeled nucleic acid probe based on the principle of nano gold aggregation.DNA probe 1 was combined with nano gold to form a complex,which was used to prepare gold standard binding pad;The upper end of nitrocellulose membrane was labeled with DNA probe 2 as the detection line,and the lower end was labeled with DNA probe 3 as the quality control line,assemble the gold standard test strip.Through the selection of test strip material,optimization of experimental conditions,specificity,stability,sensitivity,reproducibility,simulated sample detection and actual sample detection,the optimization and evaluation of the preparation of test strip are realized.The optimization experiment of the strip material shows that the best combination effect can be achieved by choosing JY-BX 115 glass fiber as the sample pad and binding pad of the strip,NC-a102 nitrocellulose film as the reaction film of the strip,H5072 absorbent paper as the absorption pad of the strip.The results show that 7.2 is the best pH value for DNA probe 1 to modify gold nanoparticle solution,and 30?L 10?m DNA probe 1 is the best for gold nanoparticle probe coupling under the best pH value.The UV crosslinking method was selected.3?L/cm is the most suitable concentration for the detection line.The detection results of artificial oligonucleotide samples showed that under the optimal conditions,the detection limit of nucleic acid strip for detection of Vibrio splendidus enzyme cutting solution was 4ng/ml.The results showed that under the optimal conditions,the minimum detection limit of DNA test strip for Vibrio splendidus was 8ng/ml,and the minimum detection limit of conventional PCR electrophoresis for Vibrio splendidus was 73ng/ml,it is proved that the sensitivity of the nucleic acid strip prepared by us is higher than PCR electrophoresis.Five kinds of Common Aquatic Pathogenic bacteria were used to evaluate the specificity of the nucleic acid test strip.The results showed that the specificity of the nucleic acid test strip established in this experiment was good.All the results of the reproducibility experiment are consistent,which shows that the reproducibility of the nucleic acid test strip established in this experiment is good.The stability test results showed that the nucleic acid test strip established in this experiment should be preserved at 4?,and the validity period was half of year. |
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