Rapid Detection Methods For Salmonella Based On Ferroferric Oxide Photothermal Effect Of Photonic PCR

As one of the foodborne pathogens,Salmonella is a serious threat to global public health.Rapid screening of low infective doses of Salmonella in potentially contaminated food is the key to preventing food poisoning.Traditional Salmonella detection methods have high accuracy,but they are time-consuming and laborious,while the immunochromatographic strip method is quick and simple,but the sensitivity needs to be improved.Currently,polymerase chain reaction（PCR）is used to detect bacteria,traditional PCR has a long thermal cycle time and depends on the equipment.Photonic PCR using the photothermal effect of nanomaterials shows a good application prospect.At present,ferroferric oxide（Fe₃O₄）is used extensively in photothermal therapy in conjunction with other materials,but no reports of its use as a photonic PCR for the detection of bacterial pathogens in food have been recovered.Most photonic PCR results relied on agarose gel electrophoresis,which cannot meet the needs of point of care testing.In this thesis,two methods for rapid detection of Salmonella Typhimurium（S.Typhimurium）were developed by means of the photonic PCR,immunomagnetic separation and gold nanoparticles lateral flow immunochromatography assay（LFIA）:one was photonic PCR based on the magnetic and photothermal effect of Fe₃O₄fluorescent detection,the other was photonic PCR-LFIA visual detection.The performance of the methods was analyzed to realize sensitive,simple and rapid detection of S.Typhimurium.The thesis was mainly divided into three contents,which were as follows:In the first chapter,the summary of the hazard and detection methods of Salmonella,as well as the rapid detection technology of photonic PCR based on various nanomaterials.The development of immunological detection for Salmonella and rapid detection based on photonic PCR were mainly introduced.In the second chapter,a rapid detection method of S.Typhimurium by photonic PCR based on the photothermal effect of Fe₃O₄and immunomagnetic separation was established,and compared with other rapid detection methods.The Fe₃O₄immunoprobe was successfully fabricated by coating the surface of Fe₃O₄with anti-Salmonella Typhimurium antibodies and blocking with BSA.The Fe₃O₄immunoprobe was incubated with S.Typhimurium,and the magnetic properties of Fe₃O₄were used for magnetic separation to achieve the effect of bacterial isolation and enrichment.After removing the supernatant,the reagents required for photonic PCR were added.The reaction mixture was irradiated with a laser and the nucleic acid amplification was completed by thermal cycling using the photothermal effect of Fe₃O₄.The fluorescence intensity of PCR product was measured to achieve a quantitative detection of S.Typhimurium in the sample.The detection conditions and performance of the established method were optimized and analyzed.The results were as follows:The particle size of Fe₃O₄was200³00 nm,the concentration was 10 mg·m L^-1;the denaturation temperature of the photonic PCR program was 80⁸5℃,the denaturation time was 5 s,the laser power was 0.40 W;the annealing temperature was 55⁶0℃,the annealing time was 10 s,the laser power was 0.13 W;during extension of PCR period,the laser power was 0.20 W,the number of thermal cycling rounds was 40 and the amount of fluorescent dye added was 14μL.The limit of detection for S.Typhimurium was calculated to be 36 CFU·m L^-1according to the standard curve Y=787.4X–1581（R²=0.9599）under optimal conditions.The newly established photonic PCR detection method has shown high specificity and good reproducibility.Samples of tap water,fruit juice,pork,egg whites and milk showed spiked recoveries ranging from 82.9%to 114%.In the third chapter,a visual detection method of S.Typhimurium by photonic PCR-LFIA was established,and compared with other rapid detection methods.By combining photonic PCR with colloidal gold LFIA,a visual detection based on photonic PCR can be realized.By applying the photothermal properties of colloidal gold,not only the quantitative detection of target bacteria can be realized,but also the sensitivity can be improved due to the amplification of photothermal signals.Firstly,the positive and negative primers used for photonic PCR were labeled with digoxin and biotin respectively,and the PCR products were stored for subsequent use.The gold was coated with anti-digoxin antibodies and blocked with BSA to obtain gold immunoprobe.After lateral flow test strips were assembled,streptavidin was dispensed onto the nitrocellulose（NC）membrane to serve as the test line（T-line）,and goat anti-mouse antibodies were dispensed onto the NC membrane to serve as the control line（C-line）.PCR products were mixed with gold immunoprobe and then added to the sample pads of test strips.The visual detection was realized by observing the color of the T-line and C-line.The temperature change（ΔT）in the T-line region of the strip was calculated and the standard curve was established with the concentrations of S.Typhimurium to achieve quantitative detection.The detection conditions and performance of the established method were optimized and analyzed.The results were as follows:The type of sample pads was Fusion3,the optimal p H of colloidal gold coupled with antibodies was 7.0,the optimal volume of anti-digoxin antibodies was 1.4μL,the concentration of BSA was 6%,the volume of positive and negative primers was 0.9μL,the dilution of PCR product was 40 times,the volume of gold immunoprobe was 8μL,the reaction time was 10 min.The concentration of streptavidin was 1 mg·m L^-1,and the irradiation time was 30 s.Under the optimal condition,the standard curve was Y=2.586X+2.508（R²=0.9573）,the lowest limit of photothermal detection was calculated to be 19 CFU·m L^-1.The new assay showed high specificity,repeatability and stability,the spiked recoveries were 81.0%¹09%in the actual samples.In this thesis,photonic PCR was constructed based on the photothermal effect of Fe₃O₄,the visual and photothermal detection methods of S.Typhimurium were developed by combining photonic PCR with lateral flow immunochromatography assay.This thesis optimized the program of photonic PCR,the conditions for the preparation of nanomaterial immunoprobe and affecting the detection of the methods,and the rapid accurate and sensitive detection of S.Typhimurium was realized.