基于纳米免疫磁珠快速富集4种海洋致病性弧菌的研究

RAPID ENRICHMENT OF FOUR COMMON PATHOGENIC VIBRIO IN THE OCEAN WITH IMMUNOMAGNETIC NANOBEADS

为研究海洋中常见致病性弧菌的快速分离富集方法——免疫磁珠法,本文以副溶血弧菌、哈维氏弧菌、创伤弧菌、灿烂弧菌为研究对象,制备纳米免疫磁珠,并通过一系列优化条件,用3M试纸计算捕获率来确定免疫磁珠最佳捕获条件。最后分别对4种致病性弧菌的不同菌液稀释浓度、对1株目标菌和8株非目标菌、4种混合菌进行捕获,从而评价免疫磁珠的灵敏度、特异性以及抗干扰能力。结果表明,4种磁珠偶联最佳抗体量为300、600、150、150μg。最佳捕获条件:副溶血弧菌免疫磁珠用量3mg,免疫反应45min,磁分离3min,p H 7.4缓冲液体系。哈维氏弧菌免疫磁珠用量3mg,免疫反应45min,磁分离1min,p H 6.0缓冲液体系。创伤弧菌免疫磁珠用量12mg,免疫反应60min,磁分离5min,p H 8.0缓冲液体系。灿烂弧菌免疫磁珠用量6mg,免疫反应30min,磁分离3min,p H 7.4缓冲液体系。在纯培养情况下,4种免疫磁珠灵敏度均达到10 CFU/m L数量级;分别对9株菌进行捕获,对目标菌的捕获率均达到87%以上,对非目标菌的捕获率均低于32%;对4种混合菌进行捕获时,免疫磁珠捕获率不受其它杂菌干扰。本研究证明了该免疫磁珠有较好的灵敏度、特异性和抗干扰能力,且与其它增菌方法相比,大大缩短了时间,具有较好的应用前景。

To develop a quick separation and enrichment of pathogenic vibrio in the ocean with immunomagnetic nanobeads（IMBs）, for which four vibrios（Vibrio parahaemolyticus, Vibrio harveyi, Vibrio vulnificus, and Vibrio splendidus）were tested. We evaluated the sensitivity, specificity, and anti-interference ability of each IMB made from these vibrios. Results show that for the four types of IMBs, the optimal amount of antibody conjugate was 300, 600, 150, and 150μg, respectively. The optimum capturing conditions were, in the turns of IMS amount（in mg） / immunoreaction（in min） / separation（in min） / pH： 3/45/3/7.4 for V. parahaemolyticus, 3/45/1/6.0 for V. harveyi, 12/60/5/8.0 for V. vulnificus, and 6/30/3/7.4 for V. splendidus. In pure culture, the capture limit could reach 10^0 CFU/m L for all four IMBs. In our test on nine bacteria, the capture rate on target bacteria could reach 87% and that on non-target bacteria was below 32%. In addition, the capture rate by IMB of the four vibrios mixed was not affected by other bacteria. Therefore, the IMBs have good sensitivity, specificity, and anti-interference ability, and can reduce detection time considerably, showing good application prospects.