Graduate School, Hebei North University, Zhangjiakou 075000, Hebei, China;Department of Clinical Laboratory, Hebei General Hospital, Shijiazhuang 050051, Hebei, China;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.
National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China.
Biomed Environ Sci. 2024 Apr 20;37(4):387-398. doi: 10.3967/bes2024.043.
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 (SA), (PA), and (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.
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.
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 ( < 0.05).
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.
重组酶辅助聚合酶链反应（RAP）是一种敏感的、单管、两阶段核酸扩增方法。本研究旨在开发一种基于重组人甘露聚糖结合凝集素蛋白（M1 蛋白）偶联磁珠（M1 珠）对病原体进行富集，结合 RAP 的方法，用于早期诊断三种血流感染菌（SA）、（PA）和（AB）引起的菌血症。
使用重组质粒评估检测方法的灵敏度。采用常见的血液流感菌进行特异性检测。用 M1 珠对模拟和临床血浆样本进行富集，然后进行多重重组酶辅助 PCR（M-RAP）和定量 PCR（qPCR）检测。用 Kappa 分析评估两种检测方法的一致性。
M-RAP 方法对 SA、PA 和 AB 质粒的检测灵敏度分别为 1、10 和 1 拷贝/μL，与其他细菌无交叉反应。M-RAP 检测法在 4 小时内可在血液中检测到<10 CFU/mL 的病原体，灵敏度高于 qPCR。M-RAP 和 qPCR 对 SA、PA 和 AB 的 Kappa 值分别为 0.839、0.815 和 0.856（<0.05）。
本研究开发了一种基于 M1 珠富集的血液样本中 SA、PA 和 AB 的 M-RAP 检测方法，可用于早期菌血症的检测。
