利用核酸适配体的竞争置换作用检测溶藻弧菌(Vibrio alginolyticus)

溶藻弧菌(Vibrio alginolyticus)是水产养殖中致病性较强的一种条件致病菌,为更灵敏、高效地对其进行检测,研发出一种基于核酸适配体竞争置换作用的检测方法。首先设计并制备出磁珠-核酸适配体-荧光报告探针的检测复合物(MAP检测复合物),然后利用溶藻弧菌与其核酸适配体有较好的亲和特异性,在对样品进行检测时,样品中的溶藻弧菌就会竞争MAP检测复合物中的核酸适配体,从而置换出与该核酸适配体结合的荧光报告探针,再通过检测置换出的报告探针的荧光强度来表征溶藻弧菌的浓度,从而实现对溶藻弧菌的定量检测。结果表明,该方法对溶藻弧菌的检测限可达到1CFU/mL,与嗜水气单胞菌(Aeromonas hydrophila)、迟钝爱德华氏菌(Edwardsiella tarda)、哈维氏弧菌(Vibrio harveyi)、大肠杆菌(Escherichia coli)等水产养殖中的常见致病微生物没有交叉反应,并在1~20、20~100和100~1000 CFU/mL范围内都表现出较好的线性关系。另外还对MAP检测复合物的制备条件进行了优化,较为理想的制备条件为:100nmol/L核酸适配体与100nmol/L荧光报告探针按体积比1︰1混合结合30 min,制备出50 nmol/L的核酸适配体-荧光报告探针复合物,然后该复合物再与25μg/mL的磁珠按体积比1︰1混合结合60 min,制备出相应的MAP检测复合物。利用核酸适配体的竞争置换作用检测溶藻弧菌有较好的灵敏度和特异性,展现了较好的应用前景。

DETECTION OF VIBRIO ALGINOLYTICUS BASED ON COMPETITION AND SUBSTITUTION REACTIONS RELATED TO APTAMER

Vibrio alginolyticus is an opportunistic pathogen with stronger pathogenicity in aquaculture.A new detection method based on the competition and substitution reactions related to aptamer was developed for more sensitive and efficient detection of the pathogen.A detection complex composed of magnetic bead,aptamer,and fluorescent reporter probe(MAP detection complex)was designed and prepared at first.Because of the good affinity and specificity between Vibrio alginolyticus and its aptamer,V.alginolyticus in the tested sample would compete for aptamer in the MAP detection complex,and replace the fluorescent reporter probe bound with the aptamer.The concentration of the bacterium could be reflected by the fluorescence intensity of the replaced fluorescent reporter probe,and thus the quantitative detection of V.alginolyticus was realized.The results show that the present method could detect as low as to 1 CFU/mL of V.alginolyticus.Moreover,the method had no cross-reaction with other common pathogenic microorganisms in aquaculture such as Aeromonas hydrophila,Edwardsiella tarda,Vibrio harveyi,and Escherichia coli.Good linearity was found in the range of 1~20,20~100 and 100~1000 CFU/mL of V.alginolyticus.The optimal preparation conditions of the MAP detection complex were realized in the following procedures.The aptamer-fluorescent reporter probe(AP)complex of 50 nmol/L was prepared by mixing 100 nmol/L aptamer with 100 nmol/L fluorescent reporter probe at the volume ratio of 1︰1 for 30 min,and then the 50 nmol/L AP complex was mixed with 25μg/mL magnetic bead at the volume ratio of 1︰1 for 60 min and the corresponding MAP detection complex was prepared.The detection of V.alginolyticus based on the competition and substitution reactions related to the aptamer showed better sensitivity and specificity with a good application prospect.