| Nucleic acids are important and fundamental to the development of every organism.Nucleic acid based detection methods such as Polymerase chain reaction（PCR）have been widely used in food safety,in vitro diagnosis,biological research,forensic identification,environmental monitoring and other fields.These techniques have a low tolerance for impurities,and the reliability of experimental results is limited by the extraction of highpurity DNA from complex samples.At present,nucleic acid detection is developing in a fast and convenient direction.However,nucleic acid extraction steps are cumbersome,time-consuming,low-sensitivity,high-cost,require organic solvents,have potential hazards to people and the environment,and cannot achieve instant detection（Point-of-Care Testing,POCT）and other limitations restrict the rapid detection of nucleic acids.Therefore,a novel,simple and rapid nucleic acid extraction and detection method that could improve the reaction sensitivity is urgently needed.An ideal extraction method should be able to rapidly extract and purify nucleic acids from complex samples and be compatible with downstream analysis to reduce nucleic acid loss and increase reaction sensitivity.Chitosan can realize the adsorption and dissociation of nucleic acid by changing the p H of the solution;diatomite is a low-cost natural silica material with strong adsorption capacity and self-sedimentation ability,which can extract nucleic acid from samples separation.Therefore,an ultra-fast and visualized single-tube nucleic acid detection method based on chitosan-functionalized diatomaceous earth was proposed in this paper.After optimizing the reaction conditions,it was determined that the modified diatomite could rapidly bind nucleic acid within 3 min under the condition of p H=5.0 MES buffer.Under the action of gravity,the DNA-enriched modified diatomaceous earth settled to the bottom of the tube,and the precipitate can be directly used for subsequent amplification,realizing nucleic acid detection in a single tube,without any elution steps,reducing nucleic acid loss,improving the sensitivity of the reaction.Combined with Accelerated PCR（AC-PCR）,the whole process can be completed within17 min and the results can be directly observed with the naked eye under ultraviolet light.Compared with the existing detection methods（which often take 1 h or more）,the extraction steps are simple,the detection efficiency is greatly improved,the time and cost are saved,and the reaction sensitivity is high.Different samples were detected based on the above methods.The detection of Salmonella was first performed with a detection limit of 1 CFU/m L,which was 100 times more sensitive than the conventional kit extraction method without nucleic acid enrichment.In addition,the method also showed high specificity and sensitivity for various spiked samples,and the effect was comparable to that of commercially available kits,meanwhile,the single extraction cost was only $0.001,for low resource area detection provides a new solution,for Salmonella and other food-borne pathogens of real-time detection provides a promising tool.Finally,nucleic acids of novel Coronavirus pseudoviruses in swabs and serum samples was detected,and the sensitivity was comparable to that of commercial kits.For the detection of ribonucleic acid（RNA）,the solution needs to be shaken evenly during the reverse transcription process presumably because the modified diatomaceous earth prevents the combination of RNA and reverse transcriptase at low temperature,therefore,this method is more suitable for the detection of DNA.In conclusion,this paper proposes a new rapid detection method that integrates nucleic acid extraction and amplification,which enables direct molecular diagnosis,greatly reduces labor-intensive detection steps,and is highly compatible with real-time PCR instruments.It has field applicability and can be used in various fields,including food safety testing,forensic analysis and clinical diagnosis. |
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