副溶血性弧菌磁性试纸条层析体系的优化

Optimization of chromatographic system of magnetic test strip for Vibrio parahaemolyticus

为能够快速检测水产品中副溶血性弧菌（Vibrio parahaemolyticus）,本研究首先制备了副溶血性弧菌多克隆抗体,然后将其标记在磁珠上形成特异性磁纳米探针,再分别以副溶血性弧菌多克隆抗体和山羊抗兔Ig G作为检测线T线和质控线C线,建立了双抗夹心模式的磁性免疫层析试纸条。利用正交试验设计的方法,从Tween-20、BSA、蔗糖3种因素对层析体进行优化分析,结果表明这3个因素对层析检测的影响效果为Tween-20〉BSA〉蔗糖;当Tween-20含量为5%（V/V）,BSA浓度为0.2 mg/m L,蔗糖浓度为0 mg/m L时,阳性信号最强,检测的准确性和灵敏度达到最优,通过单纯的肉眼观察,可在5～10 min实现1.6×10^4CFU/m L副溶血性弧菌的检测,通过T线所捕获磁信号的定量检测,可在30～40 min内实现4.1×10^3CFU/m L副溶血性弧菌的检测,为今后食源性致病菌的快速鉴定、诊断和检测提供了一种新途径。

To establish a rapid method for detection of Vibrio parahaemolyticus in aquatic products,a magnetic immunochromatographic test strip（ MITS） was developed. Anti-V. parahaemolyticus polyclonal antibody（ PAb） was prepared and conjugated with magnetic beads to prepare immunomagnetic beads（ IMBs）. Next,PAb and goat anti-rabbit Ig G were used as a test line（ T line） and a control line（ C line） to develop a sandwich MITS. In order to improve the accuracy and sensitivity of the test strip,the concentration of surfactant,protein and saccharides in the chromatographic system was optimized by orthogonal test. Our results showed that the effect of these three factors was Tween-20 〉BSA 〉sucrose,with the optimized chromatographic system（ 5% Tween-20,0 mg / L sucrose,0. 2 mg / m L BSA）,the MITS was capable to detect1. 6 × 10^4 CFU / m L of V. parahaemolyticus in 5- 10 min qualitatively and 4. 1 × 10^3 CFU / m L in 30- 40 min quantitatively. Our study could provide a new rapid identification,diagnosis and detection for food pathogen.