Research On Isothermal Nucleic Acid Amplification Method To Detect Listeria Monocytogenes

The gram-positive bacterium Listeria monocytogenes is a foodborne pathogen of global concern,which poses significant health problem to both human and domestic animals.Despite the great efforts to decrease its incidence,L.monocytogenes is still an important cause of listeriosis and could even cause large numbers of illnesses and deaths.L.monocytogenes is ubiquitous in nature and easily contaminates vegetables,fruits,dairy products,meat and seafood,which significantly increases the risk of food poison.Therefore,developing a rapid,specific and simple detection method of L.monocytogenes is of significant importance to food safety and human health.In the second chapter,a LAMP method for the detection of Listeria monocytogenes was constructed.Firstly,temperature,primer sequence,number of primers,concentration of internal primer and bovine serum albumin were optimized.Then,the feasibility of LAMP to detect L.monocytogenes genome DNA,culture fluids and bacteria colony were verified.The results of real-time fluorescence and agarose gel electrophoresis were used to determine the sensitivity of the experimental scheme.The targets were the different concentrations of L.monocytogenes genomic DNA.Besides,the method displayed good specificity and strong anti-jamming capacity by both fluorescence and colorimetric detection method.The LAMP method established in this chapter can be used for fluorescence qualitative detection of L.monocytogenes,and its colorimetric detection can be used for rapid detection in the field.In the third chapter,a denaturation bubble-mediated strand exchange amplification(SEA)method to detect L.monocytogenes was developed.SEA is a novel nucleic acid amplification method that only requires one pair of primers and anenzyme to detect RNA.The specie-specific primers were designed by targeting the16 S r RNA gene and the amplification reaction was performed as short as 60 min at61℃.Notably,SEA method could not only detect genomic DNA but also detect RNA by one step without requiring extra reverse transcription.The result could be visualized by naked eyes so that water bath pot would be the only equipment needed.Moreover,culture fluids and bacteria colony could be successfully detected and the method displayed good specificity and strong anti-jamming capacity.These features greatly simplified the operating procedure and made SEA method be potential for developing point-of-care testing(POCT)devices to detect viable L.monocytogenes.To sum up,two isothermal nucleic acid amplification methods were established for the detection of L.monocytogenes.The LAMP method has a low detection limit and can be used for fluorescence qualitative and visual colorimetric detection of L.monocytogenes.While,SEA method can detect viable L.monocytogenes.The reaction system is simple,and requires only a pair of primers and an enzyme.Besides,SEA could realize one step detection of RNA by one step at constant reaction.This method could also achieve fluorescence quantitative detection within a certain concentration range of the targets and visual colorimetric detection with naked eye.Therefore,the SEA method can be carried out under the condition of quick field detection without large instrument and can be used for developing POCT and chip laboratory equipment to detect viable L.monocytogenes.