Optimization And Application Of Loop-mediated Isothermal Amplification Technology In Rapid Detection Of Common Pathogenic Microorganisms In Rural Toilets

Due to its relatively poor sanitary environment,rural toilets are prone to breed a large number of pathogenic microorganisms,posing a threat to the health of residents.Therefore,it is very necessary to establish a fast and effective detection method for pathogenic microorganisms in rural toilets.In recent years,loop-mediated isothermal amplification（LAMP）technology has gained widespread attention as a rapid,simple and stable rapid detection method for pathogenic microorganisms.However,the conventional LAMP reaction can only detect one target gene at a time,the detection efficiency is low,and false positive results are prone to be produced,which makes the practical application of this technology face greater difficulties.Therefore,this article designed LAMP primers for the target genes of common pathogenic microorganisms（Staphylococcus aureus,Salmonella,Shigella）in rural toilets,and established a single LAMP detection method through primer screening;In comparison experiments,a triple LAMP reaction system was established,which can detect three pathogenic microorganisms at the same time;the use of ethidium bromide azide（EMA）reduces the impact of dead bacteria on the detection results and improves the specificity and sensitivity of the detection process;The effectiveness of this method is confirmed in the detection of water samples and actual water samples.The main findings of this article are as follows:（1）The genomic DNA of three pathogenic microorganisms including Staphylococcus aureus,Salmonella and Shigella was extracted by magnetic bead method.The OD260/OD280 were 1.73,1.78,1.82,respectively.The agarose gel electrophoresis showed uniform bands.,The extraction effect is good.（2）LAMP primers were designed for the nuc gene of Staphylococcus aureus,Salmonella inv A gene,and Shigella iap H gene.Single LAMP reaction systems were established through primer screening,and the reaction system was determined to be:10μmol/L FIP and BIP,10μmol/L F3 and B3,5μmol/L Loop F and Loop B,10×LAMP buffer（200mmol/L Tris-HCl,100mmol/L KCl,100 mmol/L（NH₄）₂SO₄,1.0%Tritonx-100）,1.0mmol/L d NTPs,0.8mmol/L Betaine,8U Bst DNA polymerase,2μL DNA template,reaction p H 8.8,Mg²⁺concentration 4mmol/L,reaction temperature65℃,the reaction time is 60min.The experimental results show that the designed three sets of primers have good specificity.The detection limits of the three pathogenic microorganisms are 7.5pg/μL（10 times better than PCR method）,7.2pg/μL（10 times better than PCR method）and 52pg/μL（equivalent to PCR method）.（3）Through the concentration ratio experiment of the three sets of primers,it was determined that the concentration of each set of primers in the triple LAMP reaction system was 1/2 of the original concentration（1.5μmol/L）,and thus the triple LAMP reaction system was established.The reaction temperature,d NTPs concentration,and Mg²⁺concentration of the system were optimized.The reaction temperature was determined to be 65℃,d NTPs concentration was 1.0mmol/L,and Mg2+concentration was 4mmol/L.The triple LAMP system has good specificity and the same sensitivity as single LAMP.（4）By comparing two commonly used fluorescent dyes in the LAMP method:SYBR Green I and hydroxynaphthol blue（HNB）,SYBR Green I was determined as the visualization dye of the triple LAMP detection system.Using ethidium bromide（EMA）to pre-process the samples can effectively avoid the influence of dead bacteria on the reaction results.Through optimization experiments,it is determined that the concentration of EMA is 1μg/m L,and the concentration of the processed sample matrix should not exceed 10⁵CFU/m L.Incubate The time is not less than 5min.（5）The test results of the simulated samples and the samples of the rural toilets actually collected show that the simulated samples containing the target pathogen can be detected,the method is effective and feasible,and the detection time is 90 minutes.