Aptamer Magnetic Microspheres Combined With Multiplex Pcr For Detection Of Listeria Monocytogenes

Aptamer magnetic microspheres are magnetic beads coated by specific aptamer,which can separate and enrich target molecules by the specific reaction of the aptamer-target material.It is simple and sensitive.Multiplex polymerase chain reaction?Multiplex PCR?amplifies several different DNA sequences simultaneously in one reaction system by adding more than one pairs of primers.It has many characteristics,such as short time cost,high sensitivity,good specificity.With the advantages of aptamer-magnetic microspheres and multiplex PCR,one rapid and accurate detection method of Listeria monocytogenes could be developed.The aptamers against Listeria monocytogenes had been screened previously.In this report,aptamer was immobilized on the surface of magnetic microsphere to preparation of the aptamer magnetic microspheres to capture Listeria monocytogenes.Based on this enrichment,multiplex PCR was developed to rapidly and accuratly detect Listeria monocytogenes.The main research results were as followed.1.Preparation of potystyrene/acrylamide magnetic microspheresFe₃O₄ nanoparticles were synthesized by coprecipitation.Oleic acid was the best dispersant in synthesis process compared with sodium citrate,PEG-20000 and PEG-4000.When ammonia?25 mL?mixed with oleic acid?3 mL?and kept reacting for 1h,magnetic nanoparticles were better dispersion.Finally,polystyrene/acrylamide magnetic microspheres were prepared by emulsion polymerization.The characteristic of Fe₃O₄ nanoparticles,oleic acid modified magnetic microspheres,and polystyrene/acrylamide magnetic microspheres,was measured by TEM,XRD,FT-IR,VSM.Magnetic microspheres were single phase magnetite and cubic crystal system.The saturation magnetization of polystyrene/acrylamide magnetic microsphere was 49.5 emu/g and all the particles were superparamagnetism particles.The amino content on the surface of polystyrene/acrylamide magnetic microsphere measured by conductometric titration method was 2.14 mmol/g,which was the functional group to link streptavidin.2.Aptamer magnetic microspheres enriched Listeria monocytogenesStreptavidin magnetic microspheres were made by covalent bonding of streptavidin and polystyrene/acrylamide magnetic microspheres.According to the results of competitive enzyme-linked ligand sorbent assay,the binding capacity of free biotin and biotin-ssDNA with magnetic nanoparticles were 7.58 nmol/mg and 0.14 nmol/mg,respectively.Then,biotined aptamer against Listeria monocytogenes was connected to magnetic microspheres called aptamer magnetic microspheres.The capture efficiency of aptamer magnetic microspheres was 65%when aptamer magnetic microspheres?0.05 mg?were incubated for 150 min in Listeria monocytogenes solution.3.Aptamer magnetic microspheres separation combined with multiplex PCR for detection of Listeria monocytogenesTwo pairs of primers iap6 and hlyl respectively amplifying segment of Listeria spp.and Listeria monocytogenes were screened to use in multiplex PCR amplification.Multiplex PCR for Listeria monocytogenes detection was developed in the optimize condition including DNA extraction method,the amount of primers and annealing temperature.In order to reduce the false positive results,PMA was added in the bacterial solution to specifically extract the DNA of living cells.After foods were contamined with Listeria monocytogenes?102 CFU,103 CFU?,the detection time of multiplex PCR combined with magnetic beads less than multiplex PCR.These results showed the method developed in our report could shorten detection time.