| 摘要: |
| 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, pH 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, pH 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, pH 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, pH 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/mL 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。 |
| 关键词: 纳米免疫磁珠 致病性弧菌 富集 捕获率 |
| DOI：10.11693/hyhz20150600167 |
| 分类号: |
| 基金项目:海洋公益性行业科研专项, 201105007 号; 宁波大学研究生科研创新基金项目, G14045 号。 |
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| RAPID ENRICHMENT OF FOUR COMMON PATHOGENIC VIBRIO IN THE OCEAN WITH IMMUNOMAGNETIC NANOBEADS |
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ZHANG Xuan, DAI Juan, WANG Zu-Zhong, ZHOU Jun, ZHANG Di-Jun, ZHANG Chun-Dan, LI Ye, SU Xiu-Rong
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School of Marine Sciences, Ningbo University, Ningbo 315211, China
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| Abstract: |
| 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 100 CFU/mL 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. |
| Key words: immunomagnetic nanobeads pathogenic vibrio enrichment capture rate |
