Mycoplasma pneumoniae (MP)is a small prokaryotic microorganism, which is a major cause of morbidity and mortality in children and adolescents. Notably, MP is commonly associated with community-acquired pneumonia (CAP) (Jain et al., 2015). In clinical practice, the typical symptoms of MP infection include fever, cough, sore throat, muscle pain, and others (Jiang et al., 2021). Additionally, bronchospasms, pneumonia, acute respiratory distress syndrome (ARDS), myocarditis, and meningitis have also been reported in severe cases (Narita, 2010). More than 12% of hospitalized children with MP infection are admitted to the ICU, and over 25% of patients with MP infection can develop severe pneumonia or extrapulmonary complications (Kutty et al., 2019; Rodman Berlot et al., 2023). As the manifestations of MP infection are non-specific and similar to those of other respiratory pathogens, such as the influenza virus, a misdiagnosis or delayed treatment poses a serious health threat to the public (Poddighe, 2018); therefore, it is unreliable to make a definite diagnosis of MP infection simply by clinical presentation. Moreover, the incubation period for MP is approximately 2–4 weeks (Principi and Esposito, 2001), and most patients remain unaware of the invasion of this pathogen for 14 days after infection, which greatly increases the risk of pathogen transmission in crowded places, such as schools. Therefore, MP causes frequent CAP outbreaks in school-aged children aged 5–15 years (Yiş et al., 2008). Therefore, rapid detection methods capable of accurately diagnosing MP early in the incubation period are critical for preventing outbreaks of MP-caused CAP.

Currently, the main diagnostic methods for MP infection include culture, serological tests, and polymerase chain reaction (PCR)-based methods (Waites et al., 2017). Culture-based methods provide a solid foundation for MP infection; however, culturing MP is time-consuming and relatively insensitive owing to its slow growth and fastidious nature (Leal et al., 2020). Regarding serological tests, reliable results are highly dependent on antibodies and exhibit high rates of false negatives (She et al., 2010). The results collected from PCR-based methods serve as the gold standard for MP identification with the requirement for specific equipment and skilled technicians, which are restricted to grassroots laboratories or point-of-care detection (Schmitt et al., 2013). To address this, several isothermal amplification techniques are developed for MP detection, such as loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA) (Cai et al., 2019; Fan et al., 2020). In the LAMP assay, the reaction can be performed at a constant temperature using simple operation and equipment, and the reaction time is shorter than that of PCR-based methods (Hong et al., 2023). However, much more effort is required to overcome the shortcomings of low specificity and false-positive results (Nagai et al., 2016).

Recently, clustered and regularly interspaced short palindromic repeats and their associated protein (CRISPR/Cas) systems have provided a novel method for pathogen diagnostics. Because collateral cleavage was discovered in some Cas proteins (Cas12, Cas13a, and Cas14), the CRISPR/Cas assay was modified for nucleic acid detection. Cas12 has cis-cleavage activity and cleaves targeted double-stranded DNA (dsDNA) by using a specific single CRISPR RNA (crRNA), and produces collateral trans-cleavage and non-specifically degrades single-stranded DNA (ssDNA) (Li et al., 2019), thereby the cleavage results of CRISPR/Cas12 can be visualized by using fluorophore and quencher-labeled ssDNA, indicating the presence of the target gene (Zhu et al., 2021). However, the sensitivity of the CRISPR system is low and it cannot be directly applied in clinical testing. Therefore, isothermal amplification technology was introduced to the CRISPR/Cas system, which yields a rapid, ultrasensitive, and specific detection method for MP (Deng et al., 2022; Li et al., 2022). Moreover, the CRISPR-Cas-Integrated LAMP has been applied for Pathogen-specific point-of-care (PoC) diagnostic tests (Atçeken et al., 2022). In current CRISPR-based methods, pre-amplification of the target gene and CRISPR-mediated detection are the two main steps. Therefore, the entire reaction consists of multiple manual operations that cannot avoid cross-contamination due to complicated diagnostic procedures. To address these drawbacks, a one-step, one-pot CRISPR-based nucleic acid detection platform (CRISPR-top) was developed (Li et al., 2021; Qiu et al., 2022). However, an appropriate platform for the identification of MP remains unknown.

In the present study, we integrated the LAMP assay with the CRISPR/Cas12b method to achieve a rapid, simple, sensitive, and specific detection method for MP detection (MP-LAMP-CRISPR/Cas12b). Our findings not only provide solid data for the reaction conditions, but also demonstrate the potential value of the present platform in resource-limited regions.

MP is one of the most common causative pathogens of CAP, accounting for 30–50% of CAP during the peak years. Early and rapid diagnostic methods are the key to guiding clinicians in their choice of antibiotics. However, the common methods for the identification of MP infection are culture separation, serological tests, and PCR, which are underutilized owing to shortcomings, such as time-intensiveness, insensitivity, high cost, requirement for precise instruments, and skilled technicians (Ratliff et al., 2014; Qin et al., 2023; Zhou et al., 2023). Consequently, the early, rapid, and accurate detection of MP is crucial for the diagnosis and rational selection of antibiotics.

Currently, isothermal amplification of nucleic acid technology is widely used for rapid pathogen detection of infectious diseases owing to its high efficiency and simplicity and plays an increasingly important role in promoting the on-site detection and control of pathogen diseases. LAMP was developed by Notomi et al. in 2000 (Notomi et al., 2000). In 2019, Arfaatabar et al. investigated a LAMP technique for the rapid detection of MP in clinical specimens, showing ‘substantial’ (κ=0.77) and ‘almost perfect’ (κ=0.86) congruence between the LAMP assay, culture, and PCR methods, respectively (Arfaatabar et al., 2019). However, the interpretation of the results of the LAMP assays was subjective. Thus, Wang et al. developed a LAMP coupled with a nanoparticle-based lateral flow biosensor (LFB) assay for the objective identification of MP (Wang et al., 2019a). In addition to LAMP, other isothermal amplification techniques have also been applied for the rapid detection of MP, such as multiple cross displacement amplification (MCDA) (Wang et al., 2019b), recombinase-aided amplification (RAA) (Xue et al., 2020), RPA (Jiang et al., 2022), and strand exchange amplification (SEA) (Yang et al., 2020). However, the amplified production of isothermal amplification methods often generates false-positive results; therefore, it is essential to control undesired non-specific amplification.

The specific merit of CRISPR-Cas systems is that they can be combined with isothermal amplification, which effectively solves the problem of false-positives. In 2022, Li et al. and Deng et al. developed the RPA-CRISPR/Cas12a and ERA/CRISPR–Cas12a assays for Mycoplasma pneumoniae detection, respectively (Deng et al., 2022; Li et al., 2022). In 2023, Jia et al. coupled the MCDA technique with a CRISPRCas12a-based biosensing system to design a novel detection platform for MP infection diagnosis and clinical application (Jia et al., 2023). Compared to Cas12a, Cas12b is ultrasensitive to mismatches between the guide RNA (gRNA) and target DNA, resulting in fewer off-target or false-positive effects and presenting a more accurate system in clinical applications (Strecker et al., 2019; Yang et al., 2023). Recently, Zhou et al. developed a CRISPR-Cas12b-based detection platform combined with RPA for MP infection that accurately distinguished MP strains from non-MP strains without cross-reactivity (Zhou et al., 2023). However, the contamination from aerosol cannot be totally avoided during the transferring of RPA products to CRISPR detection system.

The existing CRISPR-based tests for MP infection diagnosis require two separate reaction steps: (1) pre-amplification of the target nucleic acid using isothermal amplification assays and (2) detection of the resulting amplification products through CRISPR-based collateral reporter decoding. These two reaction steps require multiple manual operations and liquid handling, thus complicating the diagnostic procedures, increasing the risk of cross-contamination, and hampering their wider application in various fields.

However, application of the CRISPR system to detect MP with a one-step process has not yet been established. In this study, we established a one-step LAMP-CRISPR/Cas12b platform for MP detection. The working temperatures of Cas12b (37°C to 60°C) and LAMP (55°C to 60°C) reaction were observed to overlap (Dehghan Esmatabadi et al., 2015; Yang et al., 2016), which laid the foundation to integrate CRISPR/Cas12b with LAMP in one-pot. In the current study, the whole reaction was performed in one pot at 57°C and can be finished within 1 h. Moreover, there was no need to transfer the LAMP product to the CRISPR/Cas12b system. Therefore, our method achieved rapid detection of MP and prevented cross-contamination from multiple manual operations. As the entire assay was conducted at a constant temperature, an isothermal hot block and portable blue light equipment were sufficient for the MP-LAMP-CRISPR/Cas12b detection reaction. Moreover, the total cost of per reaction was about 10 RMB and most of the component can be pre-prepared via lyophilization. Hence, the proposed method performed efficiently in resource-limited settings. In addition, The LOD of the MP-LAMP-CRISPR/Cas12b system was 33.7 copies per reaction without any cross-reactivity with other non-MP pathogens. A preliminary evaluation using 272 clinical samples showed a sensitivity of 88.11% (126/143), specificity of 100% (129/129), and consistency of 93.75% (255/272) compared to the real-time PCR method, demonstrating that the developed method is reliable for the rapid diagnosis of MP.

In summary, we successfully established a rapid, simple, and accurate one-step method for MP detection by combining the LAMP assay and CRISPR/Cas12b methods in one-pot (MP-LAMP-CRISPR/Cas12b). These data indicate that the developed assay exhibits specificity and efficiency. Importantly, the MP-LAMP-CRISPR/Cas12b method has contributed to the extended detection of MP infections in various respiratory specimens, especially for point-of-care detection in resource-limited areas.

The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding authors.

The studies involving humans were approved by Approval Letter of Ethics Committee of First Affiliated Hospital of Xinjiang Medical University(K202403-32). The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent for participation in this study was provided by the participants’ legal guardians/next of kin.

TL: Formal analysis, Funding acquisition, Project administration, Resources, Software, Writing – original draft. QL: Conceptualization, Funding acquisition, Investigation, Methodology, Validation, Writing – original draft. FC: Methodology, Software, Writing – review & editing. YS: Data curation, Writing – review & editing. GM: Validation, Writing – original draft. ZY: Data curation, Formal analysis, Writing – review & editing. SZ: Writing – original draft. XL: Writing – review & editing. HL: Writing – review & editing. ZC: Project administration, Supervision, Visualization, Writing – review & editing.