Establishment of a Multiplex Detection Method for Common Bacteria in Blood Based on Human Mannan-Binding Lectin Protein-Conjugated Magnetic Bead Enrichment Combined with Recombinase-Aided PCR Technology  

作　　者：ZHAO Zi Jin CHEN Xiao Ping HUA Shao Wei LI Feng Yu ZHAO Meng XING Chen Hao WANG Jie TIAN Feng Yu ZHANG Rui Qing LYU Xiao Na HAN Zhi Qiang WANG Yu Xin LI Hong Yi SHEN Xin Xin MA Xue Jun TIE Yan Qing 

机构地区：[1]Graduate School,Hebei North University,Zhangjiakou 075000,Hebei,China [2]Department of Clinical Laboratory,Hebei General Hospital,Shijiazhuang 050051,Hebei,China [3]National Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases,NHC Key Laboratory of Medical Virology and Viral Diseases,National Institute for Viral Disease Control and Prevention,Chinese Center for Disease Control and Prevention,Beijing 102206,China [4]National Institute for Communicable Disease Control and Prevention,Chinese Center for Disease Control and Prevention,Beijing 102206,China [5]Department of Clinical Laboratory Pathology Diagnostic Center,Shiyan General Hospital,Shiyan 442000,Hubei,China [6]Graduate School Hebei Medical University,Shijiazhuang 050031,Hebei,China

出　　处：《Biomedical and Environmental Sciences》2024年第4期387-398,共12页生物医学与环境科学（英文版）

基　　金：funded by the National Key R&D Program of China[2021YFC2301102];National Natural Science Foundation of China[82202593];Key R&D Program of Hebei Province[223777100D].

摘　　要：Objective Recombinase-aided polymerase chain reaction(RAP)is a sensitive,single-tube,two-stage nucleic acid amplification method.This study aimed to develop an assay that can be used for the early diagnosis of three types of bacteremia caused by Staphylococcus aureus(SA),Pseudomonas aeruginosa(PA),and Acinetobacter baumannii(AB)in the bloodstream based on recombinant human mannanbinding lectin protein(M1 protein)-conjugated magnetic bead(M1 bead)enrichment of pathogens combined with RAP.Methods Recombinant plasmids were used to evaluate the assay sensitivity.Common blood influenza bacteria were used for the specific detection.Simulated and clinical plasma samples were enriched with M1 beads and then subjected to multiple recombinase-aided PCR(M-RAP)and quantitative PCR(qPCR)assays.Kappa analysis was used to evaluate the consistency between the two assays.Results The M-RAP method had sensitivity rates of 1,10,and 1 copies/μL for the detection of SA,PA,and AB plasmids,respectively,without cross-reaction to other bacterial species.The M-RAP assay obtained results for<10 CFU/mL pathogens in the blood within 4 h,with higher sensitivity than qPCR.M-RAP and qPCR for SA,PA,and AB yielded Kappa values of 0.839,0.815,and 0.856,respectively(P<0.05).Conclusion An M-RAP assay for SA,PA,and AB in blood samples utilizing M1 bead enrichment has been developed and can be potentially used for the early detection of bacteremia.

关 键 词：Staphylococcus aureus Pseudomonas aeruginosa Acinetobacter baumannii Human Mannan-binding lectin protein Bloodstream infection Recombinase-aided PCR assay Multiple detection 

分 类 号：R446.5[医药卫生—诊断学]