Preparation, preservation and application of immunomagnetic beads for detecting Escherichia coli O157:H7

Abstract:

[Background] Escherichia coli O157:H7 is the main serotype of Enterohemorrhagic Escherichia coli causing outburst of foodborne disease. [Objective] Our goal is to prepare immunomagnetic beads (IMBS) which are high efficient, stable and broad-spectrum, and also to improve the detection rate of target bacteria from food samples by combining immunomagnetic separation and molecular detection technology like loop-mediated isothermal amplification (LAMP), PCR, etc. [Methods] We used MIX&GO as activating agent of carboxyl magnetic beads, and successfully prepared immunomagnetic beads coupled with commercial polyclonal antibody. Subsequently, its universality and specificity were evaluated. The preservation solution of immumomagnetic beads consisting of bovine serum albumin, casein, trehalose, polyvinyl pyrrolidone (PVP), ascorbic acid and ProClin300, has been optimized by orthogonal experimental design L18(37). Six detecting methods including IMBS-LAMP, IMBS-PCR, IMBS-biochemistry, LAMP, PCR and biochemistry respectively without IMBS were adopted to detect E. coli O157:H7 in 20 ground raw pork meat samples. [Results] The capture efficiency of prepared immunomagnetic beads is 81.5%±1.3% when applied in the sample matrix of PBS solution, but decreasing in complex food matrix. The optimal formula for the preservation solution of the immunomagnetic beads is bovine serum albumin 15.0 g/L, casein 10.0 g/L, trehalose 10.0 g/L, PVP 2.0 g/L, ascorbic acid 5.0 g/L, and ProClin300 2.5 g/L. The results showed that the most sensitive method was the combination of IMBS and LAMP; 9 positive samples were respectively detected by self-prepared IMBS-LAMP and commercial IMBS-LAMP. But there was an inconsistency in two groups of positive samples because of different antibody sources of self-prepared and commercial IMBS. [Conclusion] Compared to commercial immunomagnetic beads, the prepared beads have good specificity and broader spectrum of E. coli O157 strains. Our study also showed that the IMBS-LAMP scheme could effectively enhanced detection rate of target bacteria. The IMBS-LAMP technique could be considered a high sensitive detection method of application prospect.

Key words:Escherichia coli O157:H7, immunomagnetic beads, LAMP, rapid detection