Studies On Detection Of Escherichia Coli O157:H7 With Aptamer Functionalized Photonic Crystal Microspheres

Escherichia coli O157:H7 is a common foodborne pathogenic bacteria,under certain conditions will produce a variety of toxins,gastrointestinal tract,kidney,lung,spleen and other damage,may lead to acute renal failure or even death,endangering people's health.At present,the detection methods of E.coli O157:H7 are complicated in operation,time consuming and expensive in instrument.So it is very important to develop a fast,simple and accurate method for the detection of E.coli O157:H7.A fast and efficient method for the detection of E.coli O157:H7 in food by using photonic crystal microspheres as carrier and aptamer as molecular recognition material was established.First,silica photonic crystal microspheres with controllable size and uniform structure were prepared by self-assembly platform in the laboratory,secondly,carboxyl functional groups were modified on the surface of photonic crystal microspheres,coupled to modify aptamers containing amino functional groups to obtain aptamer-modified photonic crystal microsphere materials,which could selectively identify and capture E.coli O157:H7,finally,the aptamer modified by Cy3 fluorescent groups was used to label the captured E.coli O157:H7.Thus,a double-aptamer sandwich method for fluorescence analysis of E.coli O157:H7 was established.The key factors in the analysis and detection process were optimized,and the optimal experimental conditions were determined that the concentration of amino-based aptamer was 300 nmol/L,the incubation time of E.coli O157:H7 amino-based aptamer and E.coli O157:H7 was 90 min,the concentration of Cy3 modified aptamer was200 nmol/L,the incubation time of E.coli O157:H7 modified aptamer and E.coli O157:H7was 60 min.The linear detection range of E.coli O157:H7 is 50-10~6CFU/mL,the lowest detection limit is 50 CFU/mL,and the linear equation is y=11867.4381 x+6451.3323(x is the lg value of E.coli O157:H7 concentration,y is the fluorescence intensity change).R~2=0.99533.The standard recovery of the established double-aptamer sandwich method was tested with pig meat,purified water and pure milk as the actual samples respectively,the overall recovery was between 73.61±3.87%?92.01±9.92%,and compared with the standard recovery rate of the traditional immunomagnetic separation method for the detection of E.coli O157:H7 in the same actual samples,the feasibility and accuracy of this method were further verified.