Ultrasensitive, Specific, and Rapid Detection of Mycoplasma pneumoniae Using the ERA/CRISPR–Cas12a Dual System
updated time：2026-01-21
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Ultrasensitive, Specific, and Rapid Detection of Mycoplasma pneumoniae Using the ERA/CRISPR–Cas12a Dual System (Impact Factor: 5.2), published in Frontiers in Microbiology, introduces a novel detection mode for Mycoplasma pneumoniae developed by the research team from Hengyang Medical School, University of South China. This model integrates the Enzymatic Recombinase Amplification (ERA) and CRISPR–Cas12a technology systems.
The core scientific value of this study lies in leveraging the efficient isothermal amplification characteristics of ERA technology and the organic integration with the precise recognition capability of CRISPR technology, which optimizes the accurate detection system. It provides robust data support and practical references for the scientific research applications of Mycoplasma pneumoniae.
Experimental Materials: Mycoplasma pneumoniae standard strain (M129, ATCC 29342) and 9 types of control strains.
Nucleic Acid Extraction: Genomic DNA of each strain and experimental sample was extracted according to the kit instructions, and stored at -80℃ for later use to ensure the integrity and stability of nucleic acids.
Standard Plasmid Construction: The 269bp conserved fragment of the Mycoplasma pneumoniae P1 gene was cloned into the pUC57 vector to construct a standard plasmid, which was then serially diluted (10⁶~10⁰ copies/μL) for subsequent sensitivity verification experiments.
ERA Rapid Amplification: A fluorescent detection system and a lateral flow strip detection system were constructed separately. Focusing on the optimization of ERA technical parameters, the concentration of ERA primers, reaction temperature and time were adjusted emphatically, and the concentration of specific recognition components was adaptively optimized to determine the optimal reaction conditions that balance amplification efficiency and recognition accuracy.
Performance Verification: The sensitivity of the system was verified using serially diluted standard plasmids; the specificity was verified with 9 types of control strains. A total of 92 experimental samples (56 positive and 36 negative, confirmed by quantitative real-time PCR (qPCR)) were selected for applicability verification. With qPCR results as the gold standard, Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) were calculated to evaluate the practical application value of the system.
Conclusion
Sensitivity: The limit of detection (LOD) of the ERA/CRISPR–Cas12a fluorescent system reached 1 copy/μL, which was comparable to that of commercial qPCR. The LOD of the lateral flow strip system was 100 copies/μL, meeting the requirements of clinical detection.
Specificity: No cross-reactivity was observed with the 9 control strains, and both the fluorescent system and the lateral flow strip system exhibited 100% specificity.
Clinical Performance: For clinical samples, the PPA and NPA of the fluorescent system were both 100%, showing complete consistency with qPCR results. The lateral flow strip system achieved a PPA of 92.86% and an NPA of 100%, exhibiting excellent clinical concordance.
Detection Speed: Corely relying on the rapid amplification capability of ERA technology, ERA amplification takes only 15 minutes, and the subsequent specificity verification takes 15-20 minutes. The total duration of the core process is only 30 minutes, highlighting the high-efficiency advantage of ERA isothermal amplification technology.
III. Advantages
ERA technology possesses the advantages of ultrasensitive detection, high specificity, rapidity and efficiency, as well as simple operation. This study successfully establishes the technical integration of "efficient amplification + precise recognition", which effectively addresses the problems of traditional detection technologies, such as insufficient sensitivity, complex operation, long time consumption, and poor scenario adaptability. In the future, efforts will be made to further optimize the stability of ERA reagents and sample processing procedures, expand its application scope to scenarios such as environmental samples and model samples, and enhance the value of technological transformation.
Doi: 10.3389/fmicb.2022.811768