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

Jin Zi Zhao; Ping Xiao Chen; Wei Shao Hua; Yu Feng LI; Meng Zhao; Hao Chen Xing; Jie Wang; Yu Feng Tian; Qing Rui Zhang; Na Xiao Lyu; Qiang Zhi Han; Xin Yu Wang; Yi Hong LI; Xin Xin Shen; Jun Xue MA; Qing Yan Tie

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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

Author: Jin Zi ZHAO ^{1
,

2
,

3} ; Ping Xiao CHEN ; Wei Shao HUA ; Yu Feng LI ; Meng ZHAO ; Hao Chen XING ; Jie WANG ; Yu Feng TIAN ; Qing Rui ZHANG ; Na Xiao LYU ; Qiang Zhi HAN ; Xin Yu WANG ; Yi Hong LI ; Xin Xin SHEN ; Jun Xue MA ; Qing Yan TIE
Author Information

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
Keywords: Staphylococcus aureus; Pseudomonas aeruginosa; Acinetobacter baumannii; Human Mannan-binding lectin protein; Bloodstream infection; Recombinase-aided PCR assay; Multiple detection
From: Biomedical and Environmental Sciences 2024;37(4):387-398
CountryChina
Language:Chinese
Abstract: 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 mannan-binding 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.