Application Of Fluorescent Biosensors Based On G-quadruplex And Rolling Circle Amplification In The Detection Of Emetic Bacillus Cereus

Bacillus cereus(B.cereus)is a kind of facultative aerobic gram-positive bacteria,widely distributed in air,soil and sewage,and various raw and cooked foods such as fried rice,milk and meat products are also the main pollutants.B.cereus can cause food poisoning and infectious diseases,and the symptoms of poisoning are mainly vomiting and diarrhea,which are related to cereulide and enterotoxin,respectively.It has been studied that cereulide are well tolerated in the environment compared to enterotoxins,and are produced in food contaminated with emetic B.cereus.Therefore,the establishment of an effective detection method is of great significance for the prevention and monitoring of poisoning incidents caused by eating food contaminated with emetic B.cereus.Rolling circle amplification(RCA)is a constant temperature nucleic acid amplification technology that combines single-stranded DNA(ss DNA)primers with circular DNA templates.The primer is extended with the template to produce a large number of long-chain DNA molecules to achieve nucleic acid signal amplification.RCA is widely used because of its constant temperature response,easy design and operation,and can be combined with different signal output strategies.In this study,based on RCA nucleic acid signal amplification technology,combined with blocker-tailed polymerase chain reaction(Bt-PCR)and aptamer technology for the detection of emetic B.cereus in milk.The contents of each chapter were as follows:Chapter one:This chapter introduced the principles of G-quadruplex and RCA technology,and reviewed the application research progress of G-quadruplex and RCA strategies in biosensors.Chapter two:In this chapter,the Bt-PCR-RCA fluorescent biosensors was established for the detection of emetic B.cereus in milk spiked sample.First,the primers were designed with the specific gene ces B of emetic B.cereus,and the Bt-PCR product obtained after PCR amplification,using the linker DNA as the“bridge”to bind the dumbbell DNA initiate the RCA assay,generating numerous repetitive G-quadruplex structures.The specific fluorescence dye of thioflavin T(Th T)could bind to the G-quadruplex structures,producing enhanced fluorescent signals for detection.Under optimized conditions,the limit of detection(LOD)of this method for emetic B.cereus in pure solution is 2.6×10~0CFU/m L.The detection limit is 2.8×10~0CFU/m L,and the method has good specificity.Chapter three:In this chapter,an Apt-RCA fluorescent biosensors has been developed for the detection of emetic B.cereus in milk spiked samples.This method synthesized DNA-functionalized magnetic beads.The aptamer can specifically bind to the target,competing for c DNA strands that are complementary to the aptamer,after magnetic separation,the c DNA existed in the supernatant,when put into the RCA circular template,c DNA strand can be hybridized as primers with circular template.In the presence of Phi29 DNA polymerase,RCA was initiated,and a large number of RCA products that can form a G-quadruplex structure are produced.After added Th T to the system,and Th T is embedded in G-quadruplex to enhance the fluorescence signal,realizing the fluorescence detection of emetic B.cereus in the sample.The feasibility of the method was verified by agarose gel electrophoresis and fluorescence assay experiments,and important experimental parameters such as the incubation time of the aptamer with the target,the concentration of circle template and the RCA reaction time were optimized.Under the optimized conditions,the LOD of emetic B.cereus in pure solution is 1.4×10~2CFU/m L,and the LOD of emetic B.cereus is 2.2×10~3CFU/m L in milk spiked sample.This method requires short detection time,simple operation and has good specificity.Chapter four:In this chapter,an Apt-ELRCA fluorescent biosensors was established for the fluorescent detection of emetic B.cereus in milk spiked samples.The aptamer-functionalized magnetic material is put into the sample.The aptamer and B.cereus specifically bind to compete for the complementary strand c DNA.After magnetic separation,the complementary strand c DNA remaining in the supernatant can be used as the primer for ELRCA reaction,achieving exponential amplification and accumulation of short chains containing G-quadruplexes.The combination of the formed G-quadruplex single-chain structure and Th T,which can generate fluorescent signals.The feasibility of the method was verified by agarose gel electrophoresis and fluorescent assay experiments,and experimental parameters such as the concentration of the RCA circle template,the concentration of the second primer,and the ELRCA reaction time were optimized.Under the optimized conditions,the LOD of emetic B.cereus in pure solution is 3.6×10~1CFU/m L,and the LOD of emetic B.cereus in milk spiked sample is 3.0×10~2CFU/m L.In summary,fluorescent biosensors were constructed based on G-quadruplex and RCA signal amplification technology combined with PCR and aptamer for the sensitive and specific detection of emetic B.cereus in milk.In addition,the fluorescent biosensors had good sensitivity and specificity,and provides a new technical reference for the rapid screening of food-borne pathogens in my country.