New Detection Methods Of Escherichia Coli Based On Hybridization Chain Reaction And Nanoflowers

Escherichia coli(E.coli)is widely distributed in nature,mainly divided into non-pathogenic and pathogenic E.coli.The typical pathogenic of E.coli is E.coli O157:H7.Once it is infected,it can cause severely impair to people.The traditional approaches used to detect for E.coli identification are time-consuming and require expensive equipment that must to be operated by trained technicians.Therefore,it is significance to establish a new rapid and simple detection method for E.coli.In recent years,the rapid development of molecular biology has led to the innovation of new detection methods of pathogenic bacteria,which provides more convenient,rapid,sensitive and specific detection methods.As a common signal output method in the field of biological monitoring,chromogenic reaction and test strip have many advantages,such as naked eye visibility,easy reading and so on.Based on the test strip and chromogenic reaction,combined with different molecular biological detection techniques,the following methods were successfully established:(1)Colorimetric detection of E.coli based on DSN and HCR signal amplificationBased on duplex specific nuclease(DSN)and hybridization chain reaction(HCR),a simple,rapid,sensitive and specific method for detection of E.coli was successfully established.The principle of detection is,the DNA strand in DNA-RNA double stranded nucleic acid of DSN enzyme was used to preserve the character of RNA strand,and the detection target was repeatedly used to amplify the signal.Combined with HCR,the effect of dual-amplification was achieved by using its isothermal amplification property.Using the test strip as the signal output method,the limit of detection(LOD)is 10 p M.Its advantage is high sensitivity,high specificity,short period and visualization.(2)Visual detection of E.coli based on strand displacement and HCR signal amplificationWe utilized the 16 S r RNA probe established the E.coli detection system.The target RNA is converted to DNA signal directly through the nucleic acid strand displacement,so that the DNA signal no need to reverse transcription.Combined with HCR and using its characteristics,E.coli detection system with signal amplification is formed,which effectively improves the sensitivity and stability of the detection.The visual test strip is used as the signal output mode and the LOD is 10 CFU/m L.(3)Hemin-incorporated nanoflowers as enzyme mimics for colorimetric detection of E.coliAs a new detection platform in the field of biological detection,nanoflowers have developed a variety of patterns,such as protein-protein nanoflowers,nucleic acid-protein nanoflowers and so on.In this study,we found that hemin-concanavalin A hybrid nanoflowers(HCH nanoflowers),as solid mimic peroxidase,could catalyze oxidation of 2,2’-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)diammonium salt(ABTS)in the presence of H2O2 to a green-colored product.Using this color reaction as a signal output,E.coli can be detected with sensitivity and specificity and the corresponding detection limits was as low as 4.1 CFU/m L.In conclusion,the new E.coli detection methods have many advantages,such as high sensitivity,high specificity,fast visualization,no need of large complex instruments,constant temperature,and so on.It will lay a foundation for the efficient and rapid detection of foodborne pathogens,especially E.coli,and will have a broad application prospect in clinical diagnosis and household use.