Study On Application Of Fluorescent And Colorimetric Biosensors Based On Rolling Circle Amplification In The Detection Of Cronobacter

Cronobacter is an important foodborne opportunistic pathogen that could cause necrotizing enterocolitis,meningitis,bacteremia,and even death.Infants and Immunocompromised patients are the majority of Cronobacter infected population.Study showed that Cronobacter contaminated powdered infant formula(PIF)is epidemiologically predominant cause of infant infections.Thus,it is important to develop an accurate and efficient detection methods for screening and control the food poisoning breaks caused by Cronobacter.Rolling circle amplification(RCA)is a nucleic acid isothermal amplification technology,by the combination between single strand DNA(ss DNA)primer and circular DNA template,the primer could extend along with the template under the action of DNA polymerase,and produce numerous long strand DNA with repeat sequences,and then achieve nucleic acid signal amplification.RCA technology has been widely used because it is isothermal reaction,simple and convenient,and could combine with different signal output strategy.In this study,three fluorescent and colorimetric sensors have been developed based on the RCA technology,ligase chain reaction and aptamer,for the detection of Cronobacter in PIF.The contents of each chapter were as follows:Chapter one:This chapter reviewed the advances on application of RCA-mediated nucleic acid signal amplification strategy in biosensors.Chapter two:In this chapter,an LCR-RCA based fluorescent and colorimetric biosensors has been developed for the detection of Cronobacter spp.in PIF.First,a pair of padlock primers based on specific ITS gene of Cronobacter spp.were designed.This pair of primers could complementary with the ITS gene in genomic DNA of Cronobacter spp.,under the action of Taq DNA polymerase and Taq DNA ligase,it could extend and cyclize,producing circular DNA molecules.Besides,the cyclized DNA molecules could act as templates for uncyclized primers to achieve LCR amplification and producing numerous LCR products.The LCR products could be used for HRCA or LRCA system to achieve fluorescent or colorimetric detection of Cronobacter spp.The agarose gel electrophoresis,fluorescent detection and absorbance detection were used to verify the feasibility of the proposed methods.under the optimized condition,the limit of detection(LOD)of the proposed methods for C.sakazakii were 8.6×10~1CFU/mL(fluorescent)and 7.5×10~2CFU/mL(colorimetric)in pure culture.the LOD of the proposed methods for C.sakazakii were 9.2×10~2CFU/mL(fluorescent)and 8.4×10~3CFU/mL(colorimetric)in PIF.The proposed methods showed good specificity in Cronobacter detection.Chapter three:In this chapter,an aptamer-RCA homogeneous fluorescent sensor has been developed for the detection of C.sakazakii in PIF.The reported specific aptamer was act as recognition element.In the homogeneous system,the aptamer could recognize and combine with target bacteria,and un-combined aptamers could act as primers for RCA template,and initiate RCA reaction,producing numerous G-quadruplex RCA products.The Thioflavin T(ThT)could combine with G-quadruplex,producing enhanced fluorescent signal,to achieve the fluorescent detection of C.sakazakii.The feasibility of the proposed methods has been verified via agarose gel electrophoresis and fluorescent detection.Some critical parameters have been optimized such as the incubation time between aptamer and target bacteria,the RCA template concentration,and RCA incubation time,and also evaluated the LOD and specificity of the proposed methods.under the optimized condition,the LOD of the proposed methods for C.sakazakii is 2.7×10~2CFU/mL in pure culture,and 2.4×10~3CFU/mL in PIF.The proposed methods could achieve rapid and convenient detection,and has good specificity.