Research Progress Of Rolling Circle Amplification In The Detection Of Listeria Monocytogenes

L.monocytogenes is a common foodborne pathogen which poses a major threat to human safety.Infection with L.monocytogenes can lead to sepsis,meningitis,enteritis and abortion.L.monocytogenes can survival in the soil,water,plants,human or animal waste,and more importantly,L.monocytogenes can still live at refrigerated temperatures,which poses a significant threat to stored food.Therefore,rapid and sensitive detection methods to effectively prevent and control food poisoning events caused by L.monocytogenes are extremely important.Rolling circle amplification(RCA)is an isothermal DNA amplification technique that generates a large amount of DNA under the action of DNA polymerase by introducing an initiation chain and a circular template.The method is simple in operation and design,the generated DNA can well combine probe or dye to realize signal output,and is widely used in biological detection.Based on the RCA reaction,this study combines asymmetric polymerase chain reaction(aPCR)and aptamer biosensors to detect L.monocytogenes in lettuce.The contents of each chapter are described as follows:The first chapter reviewed the research progress of rolling circle amplification technology in biological detection.The second chapter established a method of aPCR-RCA for detection of L.monocytogenes.Dumbbell DNA template was formed by blunt-end hairpin DNA under the action of T4 DNA ligase.When L.monocytogenes is present,the aPCR process produced a large amount of single-stranded DNA(ssDNA),and the ssDNA hybridized with the dumbbell DNA template,RCA reaction occurred after the addition of the phi29 DNA polymerase,dNTPs,and BSA,then a large amount of G-quadruplex DNA sequences were generated.Thioflavin T(THT)combined to G-quadruplex DNA sequences and a high fluorescence would be obtained.The L.monocytogenes was mionited by measuring the fluorescent signal.In this experiment,the dumbbell DNA template was verified by agarose gel,and the RCA reaction was successfully carried out.The amount of dumbbell DNA template and THT was optimized,and the sensitivity and specificity of the method were studied.Under the optimal conditions,the detection limit of the method in phosphate-buffered saline(PBS)was 4.8×10~1CFU/mL;the detection limit in lettuce was 4.0×10~2 CFU/g.The third chapter established a method based on RCA-aptamer biosensor for the detection of L.monocytogenes.Add the L.monocytogenes to the ELISA plate which contained the Biotin-probe 1 and aptamer complex(Biotin-Probe 1~aptamer).The aptamer specifically binded to L.monocytogenes,after washing away the aptamer~L.monocytogenes,Biotin-Probe 1 would be obtained in the ELISA plate.On the one hand,the Biotin-Probe 1 could directly hybrid to the Biotin-Probe 2,and combined with the subsequently added streptavidin-horseradish peroxidase(SA-HRP),catalyzed 3,3',5,5'-tetramethylbenzidine(TMB).Then a color reaction would occur,the concentration of L.monocytogenes could quantitatively by a microplate reader;on the other hand,Biotin-Probe 1 could hybrid with the RCA Probe,and RCA reaction would be occurred after added dNTPs and phi29 DNA polymerase,then a large amount ssDNA would be produced.The ssDNA could hybrid with the Biotin-Probe 3 to increase biotin loading,after binding to SA-HRP,the color reaction would be carried out,and the concentration of L.monocytogenes could quantitatively by a microplate reader.Under the optimal conditions,the limit of detection without RCA in PBS was 4.6×10~5 CFU/mL,and the limit of detection with RCA in PBS was 4.6×10~2 CFU/mL.The limit of detection with RCA in lettuce was 6.1×10~3 CFU/g.