A New Method For Rapid Nucleic Acid Extraction And Detection Of Listeria Monocytogenes

Listeria monocytogenes,as a common foodborne pathogenic microorganism,contains virulence factors that damage the human immune system and seriously threatens human health.Recently,the traditional detection methods of Listeria monocytogenes has a long time and specially instrument requirements.With the development of molecular biology,the isothermal amplification is very suitable for rapid and real-time detection because of the constant reaction temperature and the low dependence on instrument.However,the running time of the nucleic acid extraction is a major limiting to all kinds of nucleic acid detection methods.In this paper,it is the first time that we used silica membrane with chitosan modified for nucleic acid adsorption and enrichment without the participation of organic reagent in the whole process.After the optimization of the system,we proved that the adsorption efficiency of nucleic acid could be greatly improved by using 1%chitosan solution concentration with molecular weight<2 kD,binding in the MES buffer(pH=5)and eluting in the Tris buffer(pH=9).Then,a paper-based chip for rapid nucleic acid extraction and detection of Listeria monocytogenes was developed.After chitosan modified silica membrane for rapid nucleic acid extraction and SEA isothermal amplification performed in situ,visual detection macroscopic with portable UV makes detection limit up to 10~2 CFU/mL.Compared with the recent research on integrated detection,the detection time and detection limit could reach the forefront level.The rapid nucleic acid extraction and isothermal amplification detection will be completely carried out on the chip without precision instruments and the whole process can be controlled within 1 hour.As a new detection method of Listeria monocytogenes primary screening,it has great application potential.