The establishment and optimization of a Mycoplasma pneumoniae detection system based on ERA-CRISPR/Cas12a.

Abstract

Mycoplasma pneumoniae (MP) is a significant pathogen associated with community-acquired pneumonia, with considerable infectious risks posed, particularly to children and immunocompromised individuals. The current methods for detecting MP in research and clinical settings are recognized as time-consuming, instrument-dependent, and prone to non-specific cross-reactivity. Therefore, the creation of a rapid and sensitive detection method is urgently required. In this study, the MP-ERA-Cas12a system, integrating enzyme restriction amplification (ERA) with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a technology, was introduced. Three detection methods were evaluated: the two-pot system, a modified one-pot system, and a lateral flow assay (LFA) strip-based system. In the one-pot system, the amplification and detection steps were consolidated within a single reaction vessel, effectively minimizing the risk of contamination and false positives that may arise from the handling of multiple tubes. It was observed that the one-pot system generated a fluorescent signal within 1 h and produced 1.6 times higher fluorescence signal intensity compared to the two-pot system, achieving a detection limit of 1 copy/μL. In contrast, the LFA system facilitated rapid on-site screening, with visible band results appearing on the strip within 5 min of the reaction, and a detection limit of 10² copies/μL was achieved. High specificity for MP was demonstrated by all methods. Significant advantages, including rapid processing, the absence of complex instrumentation, and ease of use are offered by this detection system, making it particularly suitable for resource-limited clinical settings. The system is seen as an efficient tool for the early diagnosis of MP, with substantial public health and clinical relevance.

Importance: This study successfully combined enzyme restriction amplification (ERA) with the specific detection capabilities of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a. Based on the two-pot system established before, the one-pot system and lateral flow assay (LFA) system were developed for Mycoplasma pneumoniae (MP) detection. The MP-ERA-Cas12a system eliminates the need to open the lid during the reaction, reducing aerosol contamination, and minimizing the risk of false positives. The method does not require the use of advanced instruments or equipment and shows strong specificity while not being affected by other pathogens. As a new method of MP detection, the MP-ERA-Cas12a system has an important practical application prospect.