Study Of Fluorescence-Based Visual Nucleic Acid Detection And Its Application In Rapid Diagnosis Of Animal And Plant Pathogens

Nucleic acid detection technology is a detection method that targets nucleic acids in biology.This technology has been wildly used in food safety detection,environmental pollution monitoring,clinical diagnosis,genetic analysis,etc.From sample to result,usually,nucleic acid detection technology includes three basic steps,namely nucleic acid extraction,nucleic acid amplification,and amplicon detection.For the conventional nucleic acid detection technology,some shortcomings are still not solved yet.Some need a complex extraction and purification process,some need a time-consuming amplification step and some may need toxic gel electrophoresis.To better promote this technology in more practical situations,this subject started from the above three basic steps,targeting current defects and made some modifications and improvements.Besides,we introduced some novel molecular biology assays and built some nucleic acid analysis platforms which are visualized ? low-cost and simple.Three kinds of important pathogens that infect common agricultural products were chosen as the targets in this subject.The main research content and related conclusions of this paper are as follows:(1)To simplify the whole nucleic acid detection process as possible as we can and improve detection efficiency,this subject made several modifications in extraction.amplification and detection,respectively.First of all,we introduced the Na OH-based extraction method to rapidly get target nucleic acid from samples.This method shortened the extraction time to only 3 minutes with good nucleic acid yields;Secondly,a fast PCR assay was introduced.After further exploration and understanding of the “kinetic equilibrium”,we simplified the traditional three temperature zone PCR into two temperature zone.This could shorten the whole PCR time from 2h to less than 10 min.Then,we introduced molecular beacon technology.Basing on the research of its structure,we designed probes with high specificity and realized fluorescence visual detection with the naked eye at room temperature.Besides,by switching the group labeled at the end of the probe,we realized duplex detection in one reaction tube targeting two different pathogens.By smartly integrating those three systems,we built a wonderful platform with the features of nucleic acid fast extraction,rapid PCR amplification,end-point naked-eye detection.This platform is successfully used for the analysis of IHHNV(Infectious hypodermal and hematopoietic necrosis virus)in shrimp samples.It is also capable of field detection.This platform has a good limit of detection and can finish IHHNV analysis in 15 min comparing with 3 h by national standard methods.This improvement greatly enhances detection efficiency.(2)To better improve the specificity and sensitivity,we introduced CRISPR/Cas-based genetic editing technology.By some exploration,we found and proved a new PAM for the Cas12 a enzyme,namely “UUUN”.Based on this new PAM,we continued introducing uracil-DNA glycosylase(UDG)to help stop the contamination caused by residual amplicon.Besides,we replaced PCR reaction with loop-mediated isothermal amplification(LAMP)which is more convenient to operate and costs less reaction time.The Cirtus huanglongbing was chosen in this subject as the target which has a great impact in the orange industry.When there are target nucleic acid strands in the system,the specially designed cr RNA would couple with the target strands.These two would further form a complex with the Cas12 a enzyme.Then the bypass cleavage activity of the Cas12 a enzyme got activated,which can casually cleave the single-strand oligonucleotide in the system.Based on the property,we realized analysis for the huanglong bing with high sensitivity and specificity.After testing a bulk amount of infected samples,we built a platform with the features of anti-contamination,isothermal amplification,end-point visual detection.The platform based on the above three systems has a limit of detection at about 10copies/reaction,which is 100 times more sensitive than the traditional LAMP & dye assay.This limit of detection is nearly single copy level.The whole platform is free from contamination causing by the residual amplicon which certainly solves the trouble in the field of nucleic acid detection.(3)To further simplify the process of nucleic acid detection and improve the stability of the store of enzyme reagents,based on the results of the previous exploration,this subject intends to thoroughly eliminate the extraction and purification steps.This is aimed to realize direct amplification in whole blood samples without any pre-preparation.By comparing with three kinds of common Bst enzymes which have the ability of strand replacement.We picked up the ideal enzyme(Bst large fragment)for further experiment.Besides,to better store the LAMP reaction system and heat-sensitive CRISPR reaction system in one single tube and enhance their store stability,we explored the trehalose and pullulan-based protectant.Through the dehydration assay,the CRISPR reagent was transformed from liquid to a small solid film sheet.After testing,this film sheet can be stably stored at room temperature for about 180 days.This feature could certainly free the enzyme reagents from the need of much low temperature for store and save the cost.Also,it made much easier for long-distance traffic after shifting the cold-chain device.The subject targets the wildly influenced virus,the African swine fever virus,which resulted in a large number of losses in the pig industry.We built a visual nucleic acid analysis platform based on the dehydrated CRISPR system and free from simple extraction and purification.Using this platform,based on the fluorescence intensity at the end of the CRISPR reaction,semi-quantitative analysis of ASFV is realized.This platform has a limit of detection of 100 copies/reaction and offers a new idea to solve the problem of the extraction process of complex samples.By using sugar as protectants and dehydration operation,the enzyme reagents can be transformed from liquid to solid,this gives a new idea for better store the fragile reagents.The whole analysis process of this platform can be performed in a simple device that realizes “sample to result” detection.