Development Of Nucleic Acid Detection Methods Based On Molecular Amplification And Lateral Flow Fluorescent Immunoassay For The Detection Of Pathogenic Microorganisms

Both the human and animal kingdoms face the threat of pathogenic microorganisms,of which,bacteria and viruses are more harmful.Timely and accurate detection of pathogenic microorganisms are of great significance for scientific prevention and control.At present,polymerase chain reaction(PCR)has been extensively used in the detection of pathogenic microorganisms.However,PCR methods either rely on the observation results of agarose gel electrophoresis with safety risks,or are in need of high-cost quantitative equipment and supporting reagents.The isothermal amplification technology has made up for the shortcomings of PCR methods in point-of-care testing(POCT),but the existing methods still have some deficiencies in terms of primer design,target sequence,sensitivity,etc.,and it is difficult to achieve wide application.In addition,as an important step in nucleic acid detection,conventional DNA extraction methods for different types of samples lack versatility.The extraction steps are tedious,and the quality of the extracted DNA needs to be strengthened.In response to the above problems,this research has made improvements and innovations in nucleic acid extraction and purification,conventional PCR,and isothermal amplification methods.According to the demand of DNA/RNA detection under different application conditions,quantitative methods were established based on lateral flow fluorescent immunoassay and applied to the detection of pathogenic microorganisms.The main work is as follows:(1)Development of a universal DNA extraction and purification method based on magnetic beads.By comparing the extraction results of different magnetic beads,silico-hydroxy magnetic beads were chosen as the preferred DNA extraction magnetic beads.40μL of SDS solution(15%,w/v)was in addition to the CTAB lysate for reagent modification.Using isopropanol as the binding solution,and the preferred dosage of magnetic beads was 40μL(30 mg/m L).Finally,elution was performed at65°C.The established method could extract DNA with high quality and efficiency of various types of samples.Next,a nucleic acid purification method using magnetic beads was established.Experiments were conducted to determine the effect of the adsorption solution volume,PEG-8000 and Na Cl concentration on DNA fragments of different lengths,and successfully verified by PCR products of S.Enteritidis.(2)Establishment of a PCR quantitative assay based on lateral flow fluorescent immunoassay and its application in the detection of canine parvovirus 2.The use of fluorescent microspheres as probes for amplifying fluorescent signals increased the sensitivity of method.PCR product purification based on magnetic beads effectively solved the problem of false positives caused by primer dimers and so on,promoting accuracy of the results.The preferred working volume of the running buffer was 80μL and the immunochromatographic detection time was 2 minutes.This method could specifically detect canine parvovirus type 2 with a cutoff value of 146(fluorescence signal value)and a detection sensitivity of 30 copies/μL,which was 100 times higher than conventional PCR.The detection results of clinical samples are consistent with conventional PCR.The positive samples were analyzed by VP2 gene sequencing to determine the antigen type of the viral strains.This assay is simple,safe and fast,and the detection results can be quantitative(3)Establishment of a strand exchange amplification quantitative assay based on fluorescent lateral flow immunoassay and its application in the detection of Salmonella spp.Taking Salmonella spp.,important food-borne pathogens,as the research object,the accuracy of stand exchange amplification was verified by enzyme digestion of the products and DNA sequencing,and the amplification mechanism was analyzed.Combined with lateral flow fluorescent immunoassay,nucleic acid detection could be completed within 30 minutes and the cutoff value was 15.The sensitivity was 6 CFU/m L of Salmonella pure culture or 3×10~4CFU/25 g of artificially spiked raw chicken meat.The specificity was checked by 89 Salmonella strains and 15 non-Salmonella reference strains(different genera).236 samples were tested,which was consistent with conventional PCR.The method is simple,rapid,sensitive and specific,and suitable for rapid detection of pathogenic microorganisms.(4)Establishment of a tri-primer-enhanced strand exchange amplification quantitative assay based on lateral flow fluorescent immunoassay and its application in the detection of SARS-Co V-2.To improve the efficiency of amplification of RNA,we established a tri-primer-enhanced strand exchange amplification method.The product was verified by restriction digestion and DNA sequencing,and the amplification mechanism was analyzed.Amplification of Rd RP and N genes could be accomplished under the same reaction conditions.Results of Rd RP and N gene assays had good reproducibility,with sensitivities of 90 copies/μL and 70 copies/μL,respectively.Specificity tests showed that the novel assay can specifically detect SARS-Co V-2.Simulated viral infection experiments showed that the sensitivity of the Rd RP and N gene assays was 200 copies/m L and 90 copies/m L,and the cutoff values were 11 and 12,respectively.In summary,the universal DNA extraction method using magnetic beads provides a high-quality template for nucleic acid detection,and the PCR product purification method provides methodological support for resolving interferences such as primer dimers.The quantitative PCR method provides additional method support for the improvement of conventional PCR.The quantitative strand exchange amplification assay makes up for the shortcomings of existing methods in detecting short-sequence targets,and is suitable for POCT.Besides,the tri-primer-enhanced strand exchange amplification quantitative assay provides a novel and powerful technological support for the efficient detection of RNA pathogens.This study provides a targeted solution for rapid detection of different types of pathogens under different application conditions such as laboratories and POCT.The established methods are simple to operate,safe,rapid,sensitive,digitally quantitative and suitable for popularization,which are of important practical application value.