Shen Xinxin, Hua Shaowei, Zhao Zijin, Tian Fengyu, Li Lingjun, Ma Xuejun
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, China.
State Key Laboratory of Antiviral Drugs, NMPA Key Laboratory for Research and Evaluation of Innovative Drug, Pingyuan Laboratory, School of Chemistry and Chemical Engineering, Henan Normal University, Xinxiang City, Henan Province, China.
China CDC Wkly. 2025 Apr 4;7(14):482-488. doi: 10.46234/ccdcw2025.079.
Fluorescent probe-based recombinase aided amplification (RAA) offers the advantages of rapidity and simplicity but is limited by the requirement for complex and lengthy probe design, restricting its widespread application.
A novel EvaGreen dye-based RAA (EvaGreen-RAA) assay utilizing self-avoiding molecular recognition system (SAMRS) primers was developed for the detection of (PF) and (BC) in milk. Conventional RAA was used as a reference method. Sensitivity was evaluated using nucleic acids from recombinant plasmids and simulated milk specimens. Additionally, a dual EvaGreen-RAA assay was investigated for simultaneous detection of mixed BC and PF in simulated milk specimens.
The EvaGreen-RAA demonstrated superior sensitivity compared to conventional RAA, with detection limits of 1 copy/µL versus 10 copies/µL for both BC and PF plasmids, respectively. In simulated milk specimens, EvaGreen-RAA detected BC and PF at concentrations of 100 CFU/mL and 200 CFU/mL, respectively, compared to 400 CFU/mL and 600 CFU/mL for conventional RAA. The dual EvaGreen-RAA assay successfully detected mixed BC and PF in simulated milk specimens at concentrations of 200 CFU/mL for each pathogen.
The EvaGreen-RAA assay demonstrated significant advantages in terms of simplicity and enhanced sensitivity compared to fluorescent probe-based RAA, offering a novel approach for developing multiplex pathogen detection systems using melting curve analysis.
基于荧光探针的重组酶辅助扩增（RAA）具有快速、简便的优点，但受限于复杂且冗长的探针设计要求，限制了其广泛应用。
开发了一种基于新型EvaGreen染料的RAA（EvaGreen-RAA）检测方法，该方法利用自回避分子识别系统（SAMRS）引物检测牛奶中的（PF）和（BC）。传统RAA用作参考方法。使用来自重组质粒和模拟牛奶样本的核酸评估灵敏度。此外，还研究了一种双重EvaGreen-RAA检测方法，用于同时检测模拟牛奶样本中混合的BC和PF。
与传统RAA相比，EvaGreen-RAA显示出更高的灵敏度，BC和PF质粒的检测限分别为1拷贝/μL和10拷贝/μL，而传统RAA分别为400拷贝/μL和600拷贝/μL。在模拟牛奶样本中，EvaGreen-RAA分别在100 CFU/mL和200 CFU/mL的浓度下检测到BC和PF，而传统RAA分别为400 CFU/mL和600 CFU/mL。双重EvaGreen-RAA检测方法成功检测到模拟牛奶样本中浓度为200 CFU/mL的混合BC和PF。
与基于荧光探针的RAA相比，EvaGreen-RAA检测方法在简便性和灵敏度提高方面具有显著优势，为利用熔解曲线分析开发多重病原体检测系统提供了一种新方法。
