| Salmonella enterica is a natural existence food borne pathogen that can easily cause food poisoning.According to the statistics,the proportion of S.enterica triggered food poisoning incidents accounted for more than 70% of the total poisoning incidents caused by foodborne pathogens.S.enterica is widely distributed in fresh foods such as marine aquatic products,and these foods can't be stored for long time.Therefore,it is important to develop a rapid and accurate detection method for detecting S.enterica in food safety,disease prevention and other related fields.In order to achieve the purpose of rapid detection of Salmonella,this study has discussed and analyzed the existing Salmonella detection methods in the market,and found that only isothermal nucleic acid detection method can meet the requirements of on-site test.The second chapter studies two isothermal nucleic acid amplification methods for the detection of S.enterica.Firstly,the loop-mediated isothermal amplification was used in this study,which is regarded as the most widely applied technology in the field of isothermal nucleic acid amplification.The reaction system was optimized and applied to detect S.enterica genomic DNA.The experimental results showed that the method possessed high sensitivity.But the complex amplicon with different long fragments of DNA macromolecule is difficult to degrade in the environment,leading to aerosol pollution and resulting in false positive results in the subsequent experiments.To overcoming these shortcomings,we utilized denaturation bubble-mediated strand exchange amplification method to detect S.enterica,which was reported by our research group.It is known from the literature that this method has the disadvantages of long detection time and insufficient detection sensitivity.Therefore,this paper optimized the SEA method and realized the specific detection and anti-interference detection of S.enterica,proving the practical value of this method.However,during the study,the method also exposed the lack of sensitivity,which can only detect the genomic DNA of S.enterica with a concentration of 100 fM.It is far from meeting the requirement of national standard according to which S.enterica typhimurium should not be detected.In the follow-up experiment,this paper deeply explored the SEA method to solve the shortcoming of lack of sensitivity,so that it can satisfy the requirements of fast and accurate detection field.In the literature survey,it was found that cleaving endonuclease and DNA polymerase can greatly enhance the sensitivity of nucleic acid detection,but the combination of both enzyme will produce strong background signal interference,which is due to the cleaving endonuclease that can continuously cleave the product of the polymerase's self-reproduction.Based on this research result,we used endonuclease and polymerase to make it difficult to cut single strand in the third chapter,reducing the background signal while improving the sensitivity of detection.Because RNA can be easily degraded by ribonuclease in living organisms,it is important to detect RNA in order to determine whether organisms are living bodies.We constructed a rapid and accurate visual nucleic acid amplification detection technique to detect RNA and used it to solve social focus problem,the Zika virus.After successfully constructing this amplification technology,this study simulated polluted marine aquatic products,and carried out the detection of S.enterica in real samples.The results showed that this technology is an effective and sensitive method for nucleic acid amplification of S.enterica.Finally,the isothermal nucleic acid amplification technology can be applied to the microfluidic chip by establishing microanalysis system.This technology will avoid the possible operating errors in the manual operation,and eliminate the shortcomings such as aerosol pollution and requirement of large detection instrument,providing a new direction for the application of nucleic acid rapid detection technology. |
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