Rapid Detection Of Hypervirulent Drug-resistant Klebsiella Pneumoniae In Various Specimens Based On Magnetic Separation Combined With LAMP

Klebsiella pneumoniae（K.pneumoniae）,a gram-negative bacterium,is a clinically common opportunistic pathogen that can cause serious infections in various systems throughout the body,such as the urethra,respiratory tract,skin and soft tissue,gastrointestinal tract,blood and nervous system.In recent years,due to the innovation and abuse of antibiotics,drug-resistant strains begin to appear widely.The emergence of drug-resistant problems will directly aggravate the spread of pathogens,and the combination of high virulence and drug resistance will eventually lead to serious infection events.Therefore,it is necessary to establish a simple,rapid and sensitive method to detect hypervirulent drug-resistant Klebsiella pneumoniae,so as to improve the level of safety prediction and prevent the occurrence of infection events.However,the complex clinical sample matrix will seriously affect the sensitivity and accuracy of the detection method.The application of magnetic separation（MS）and other pretreatment techniques to the sample pretreatment process can effectively eliminate the interference of the matrix to the detection method.In the magnetic separation of pathogenic bacteria,commonly used recognition molecules mainly include antibodies,aptamers,borates,etc.Among them,immunomagnetic separation is widely used because of its high specificity.Because antigen and antibody have extremely high recognition and binding ability due to their complementary chemical structure and spatial configuration（Kd=1μmol/L～1fmol/L）.Therefore,in this study,antibody（Ab）was used as the recognition molecule,and functionalized magnetic beads（MBs）were prepared by"indirect"coupling to separate and enrich K.pneumoniae in PBS.And combined loop-mediated isothermal amplification（LAMP）to achieve sensitive and rapid detection of hypervirulent drug-resistant Klebsiella pneumoniae.The content of each chapter is as follows:Chapter 1:The research progress of MS recognition molecular types、coupling modes and the application of Ab in the detection of pathogenic microorganism was reviewed.Chapter 2:To realize the separation and detection of K.pneumoniae in the sample,a method based on MBs-Ab combined with loop-mediated isothermal amplification（LAMP）was established.K.pneumoniae was separated and enriched by"two-step"method,and the virulence gene peg-344 and resistance gene kpc-2were further amplified by loop-mediated isothermal amplification（LAMP）,and the amplified DNA underwent chromogenic reaction with SYBR Green I、HNB dye,and optimize the amount of d NTPs、Mg²⁺、Bst 2.0 and incubation temperature to achieve colorimetric detection of hypervirulent drug-resistant Klebsiella pneumoniae.Under optimal conditions,the capture efficiency（CE）of K.pneumoniae in PBS was greater than 93%,and the CE in cerebrospinal fluid and blood samples were greater than 92%and 85%.The limit of detection（LOD）in PBS、cerebrospinal fluid and blood were 2.5×10¹CFU/m L,2.5×10¹CFU/m L and 2.5×10²CFU/m L,respectively.The Immunomagnetic separation method prepared in this study has the ability to separate and enrich K.pneumoniae,and the combined LAMP can be used for sensitive and specific detection of hypervirulent drug-resistant Klebsiella pneumoniae.In conclusion,the Immunomagnetic nanomaterials prepared in this study have good enrichment and separation efficiency for K.pneumoniae in samples,and the combination of loop-mediated isothermal amplification can achieve sensitive and rapid detection of hypervirulent drug-resistant Klebsiella pneumoniae.This study can provide a reference for the monitoring of K.pneumoniae by magnetic separation technology.