基于核酸适配体的差减荧光法检测鳗弧菌(Vibrio anguillarum)

鳗弧菌(Vibrio anguillarum)可感染鳗鲡、虹鳟和大菱鲆等多种水产动物,是水产养殖中的一种重要病原菌,对其进行快速检测是病害防控的前提和基础。利用鳗弧菌与其核酸适配体之间有较强的亲和特异性,首次建立了一种基于核酸适配体的可定量检测鳗弧菌的差减荧光法。该方法对鳗弧菌有较好的特异性,对鳗弧菌的检测荧光值是其他菌(溶藻弧菌、哈维氏弧菌、铜绿假单胞菌、变形假单胞菌、嗜水气单胞菌、迟钝爱德华氏菌和大肠杆菌)的4～11倍,对鳗弧菌的最低检测限为10²CFU/mL,可用于10²～10⁸CFU/mL的范围内的定量检测。通过对不同盐度海水和鱼体组织样品进行加标回收检测,结果表明,回收率和相对标准偏差等指标均符合相应的标准,说明该检测方法可用于海水样品和水产动物组织中鳗弧菌的检测。

DETECTION OF VIBRIO ANGUILLARUM BY DIFFERENTIAL FLUORESCENCE METHOD USING APTAMER

Vibrio anguillarum infects many aquatic animals such as eel, rainbow trout, and turbot, and is an important pathogen in aquaculture industry. Rapid detection is necessary to control of V. anguillarum-induced disease. Based on the strong affinity between V. anguillarum and its aptamer, a quantitative differential fluorescence detection method of the pathogen was developed using the aptamer for the first time. The detection method is very V. anguillarum specific, and the binding fluorescence values of V. anguillarum was 4~11 times those of other bacteria such as V. alginolyticus, V. harveyi, Pseudomonas aeruginosa, P. plecoglossicida, Aeromonas hydrophila, Edwardsiella tarda, and Escherichia coli. The minimum detection limit of V. anguillarum reached 10² CFU/mL and good linear relationship was found in the detection range of 10²~10⁸ CFU/mL. Recovery experiments were carried out for the seawater samples with different salinities and different fish tissue samples. The results show that the detection indexes such as recovery rate and relative standard deviation were in line with the corresponding standards, indicating that the differential fluorescence method can be used in the detection of V. anguillarum in seawater samples and tissue samples of aquatic animals.