A Sensitive Fluorescence Quenching-Recovery Sensor Based On RCA And Hairpin MB For The Specific Analysis Of Fn.n

Generally,Fusobacterium nucleatum.nucleatum(Fn.n)is found in the oral cavity of humans and animals.But it can cause disease under certain conditions,and especially closely related to colon cancer.Traditional detection methods,such as bacterial culture and identification technology,which are considered the gold standard for bacterial detection.However,they are time-consuming,laborious and cannot meet the needs of rapid detection.New detection methods,such as polymerase chain reaction and chromatography,rely on expensive professional instruments,which are not conducive to real-time detection in the field.In this study,rolling circle amplification(RCA)is first applied to the detection of Fn.n.The specific padlock probe and capture probe were designed based on the nusG specific gene of Fn.n,At the same time,the molecular beacon(MB)technology combined with fluorescence was used instead of the traditional agarose gel electrophoresis to establish a new detection method for Fn.n.The rolling circle amplification and the fluorescence detection system were optimized respectively.The results showed that the connection temperature of the padlock probe is 67℃,the amount of Taq DNA ligase is 0.15 μL,the concentration of padlock probe is 0.4 νM,the amount of digestion product is 10 μL,the hybridization temperature of capture probe is 57 ℃,the amount of capture probe is 1.5 μL,the amount of phi29 DNA polymerase is 1 μL,the concentration of Tris solution is 0.1 M,the pH of Tris-HCl buffer is 9.0,the concentration of detection probe is 0.1 μM,and the optimal detection conditions were 72.6℃ for 40 min.Specificity analysis showed that the padlock probe had strong specificity,and no cross-react with Fusobacterium nucleatum.polymorphum,Fusobacterium nucleatum.vincentii,Bacteroides fragilis and others.The detection probe can distinguish the target sequence from the sequence with one to three base differences.Sensitivity analysis showed that fluorescence intensity increased linearly with increasing logarithm of the whole gene concentration in the range of 0.7-70,000 ng/L.The linear regression equation is F=624.93+31.50 1g C,with a correlation coefficient of 0.991.The detection limit of the detection method is 0.7 ng/L.Fecal sample spiked experiments showed that the detection method can clearly distinguish the micro-,small-,and large-scale presence of Fn.n.The detection results of food samples showed that RCA fluorescence detection method has better sensitivity and specificity in complex environments.The proposed method shows high sensitivity and specificity for the detection of Fn.n genomic DNA and can be used for rapid on-site detection,which is of great significance for the detection of Fn.n.