| Drinking water safety is an important public health issue that has attracted worldwide attention.Diseases caused by pathogenic bacteria in the water environment pose a huge threat to human health.As an important waterborne and foodborne pathogen,E.coli O157:H7 can cause diseases such as hemorrhagic diarrhea,hemorrhagic colitis,peritonitis and hemolytic uremic syndrome after infection,and even death in severe cases.It can be seen that the sensitive detection and efficient removal of the pathogenic bacteria E.coli O157:H7 are of great significance for preventing the occurrence of diseases and ensuring human health.Therefore,this paper improved the existing detection and removal methods of pathogenic bacteria E.coli O157:H7.Aiming at the detection method of Escherichia coli,based on the Recombinase Aided Amplification(RAA)and CRISPR/Cas12a technology,this paper establishes a rapid,ultra-sensitive and visual detection method for E.coli O157:H7.The current standard method for the detection of E.coli is the culture detection method.Although this method is stable and reliable,it has the problems of long detection cycle(5-7 days),cumbersome operation steps and heavy workload.Therefore,the development of a rapid,highly sensitive,highly specific and in situ detection method for E.coli is very important to ensure the safety of drinking water and food.The method proposed here utilizes the nucleic acid amplification of RAA and the specific recognition and trans-cleavage capabilities of the CRISPR/Cas12a system to achieve target amplification and signal transduction.The signal is read by the fluorescence generated by the ss DNA reporter modified at both ends of with FAM fluorescent group and the BHQ1 quencher group(ss DNA-FQ Reporter),or through the ss DNA reporter modified with the FAM fluorescent group and Biotin at both ends(ss DNA-FB Reporter)for lateral flow assay(LFA)to achieve the detection signal visible to the naked eye.The experimental results show that the method proposed in this paper can detect E.coli O157:H7 as low as～1 CFU/m L(fluorescence method)and 1x10~2CFU/m L(lateral flow assay),which greatly improves the detection sensitivity.Benefiting from the specificity of the CRISPR/Cas12a system,the method proposed in this paper also exhibits good selectivity,which can significantly distinguish target strains from non-target strains.In addition,by simulating the detection of real samples(drinking water and milk),it is proved that the method still has good applicability in real samples.Under optimized conditions,the RAA-CRISPR/Cas12a detection method established in this paper shortens the total time of the entire process(including extraction,amplification,and detection)to within 55 minutes,and the fluorescent signal can be visualized by the naked eye with a hand-held UV lamp or test strip.Because of the advantages of rapidity,high sensitivity and no complicated instrument,this method has a broad application prospect in situ detection of trace amounts of pathogens.Aiming at the removal of Escherichia coli,this paper proposes a method to prepare porous DNA/hydrogel using the natural protein-stabilized Pickering emulsion as a template,which provides a reliable candidate material for the filter material that needs to be used in the filtration removal of bacteria.At present,the eradication of Escherichia coli mainly adopts two methods of killing bacteria and removing bacteria.Traditional sterilization methods are not applicable when potable water resources need to be obtained in areas or environments where there is no relevant equipment and conditions,or when bacteria need to be removed under mild conditions.The method of removing bacteria is mainly through filtration,which does not require specific equipment or cumbersome operations,and is suitable for in-situ operations to obtain clean water sources.The efficiency of this method depends on the filter material used.Although researchers have developed many filtration materials for the removal of bacteria,there are still some limitations,such as long filtration times,low removal efficiency,high cost,and potential toxicity,which restrict the further use of filtration in drinking water treatment and other aspects.DNA hydrogel is an extremely hydrophilic three-dimensional polymer network gel material,which has the advantages of being porous,safe,green,and programmable,so it has been increasingly recognized in the fields of biomedicine and environmental engineering.However,their biocompatibility may be affected by the components used to support structure formation.Here we describe for the first time the use of natural protein stabilized Pickering emulsion as a template for the preparation of porous DNA gel.The results indicate that protein properties including isoelectric point(p I)and size have a great influence on the structure of the DNA gel.Moreover,using bovine serum albumin(BSA)as an example,the morphology and density of the pores in the gel could be well-tuned by the amounts of added proteins.Both CCK-8 and live/dead staining assay show that the as-prepared gel was highly biocompatible.The above experimental results show that the as-prepared gel can be used to prepare columns for efficiently removing multiple pollutants in water via a facile filtration process based on different retention mechanisms.This study improves the application potential of DNA hydrogels in environmental science,and at the same time provides a reliable candidate material for the filter material that needs to be used in the filtration method to remove bacteria. |
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