Pseudomonas aeruginosa (P. aeruginosa) is a Gram-negative, rod-shaped aerobe/facultative anaerobe belonging to the genus Pseudomonas of family Pseudomonadaceae. Metabolically, P. aeruginosa is versatile and can adapt to a wide range of niches, including soil, aquatic environment, plants, animals, and human beings (Silby et al., 2011; Gellatly and Hancock, 2013; Moradali et al., 2017). P. aeruginosa is an important opportunistic pathogen of human beings, especially for the vulnerable patients with cystic fibrosis (CF) lungs (Turner et al., 2015), obstructive pulmonary diseases (Eklöf et al., 2020), and other immunocompromised and hospitalized patients (Malhotra et al., 2019). It infects three-quarters of patients with CF and leads to high morbidity and mortality rate (Surette, 2014). Meanwhile, P. aeruginosa is the predominant pathogen causing otitis media (Mittal et al., 2015), keratitis (Hilliam et al., 2020), endocarditis (Sheppard, 1991), bacteremia (Fabre et al., 2019), burn and wound infections (Salerian, 2020), urinary tract infections (Yin et al., 2022), and more. However, treatment of P. aeruginosa infection in clinical setting has confronted with great challenge due to its resistance to different antibiotics and antiseptic (Lister et al., 2009; Chevalier et al., 2017). A combination of intrinsic, acquired, and adaptive ability of P. aeruginosa to counter antibiotic attack (Pang et al., 2019) and its extensive reservoirs in nosocomial and community environments (Ratnam et al., 1986; van Asperen et al., 1995; Yakupogullari et al., 2008; Quick et al., 2014) complicated the effective treatment and control of P. aeruginosa infection and rendered it a healthcare concern (Rosenthal et al., 2016). P. aeruginosa has become one of the notorious “ESKAPE” pathogens (Pendleton et al., 2013) and been considered as the “critical” category of the World Health Organization’s priority list of bacterial pathogens for which research and development of new antibiotics is urgently needed (Tacconelli et al., 2018). Under this context, development of rapid, accurate, and sensitive detection method for P. aeruginosa is of vital importance and urgently required for early diagnosis of P. aeruginosa infection and effective control its wide spread.

In clinical settings and routine laboratories, specimen culture is the most common and gold standard for P. aeruginosa identification (Rytter et al., 2020). Use of culture-based method is able to determine antibiotic susceptibility; however, obtaining the results usually takes a minimum time of 48 h, which may delay antibiotic treatment and compromise patient outcome (Rytter et al., 2020). In this regard, more rapid and sensitive diagnostic tests for P. aeruginosa detection are still urgently needed. During the past decades, a plenty of polymerase chain reaction (PCR)–based techniques, including conventional PCR and real-time PCR methods, have been widely developed and applied in pathogen identification including P. aeruginosa (Williams et al., 2010; Lim et al., 2021). Although sensitive and specific, these methods usually rely on sophisticated instruments and well-trained technicians, which commonly equipped in the well-established laboratories and normally cost more than 1 h to report results. More recently, several isothermal nucleic acid amplification techniques that overcome the limitations of PCR-based methods have been reported for P. aeruginosa detection, such as recombinase polymerase amplification (RPA) assay (Yang et al., 2021), multiple–cross-displacement amplification (MCDA) assay (Li et al., 2020; Wang et al., 2020), and loop-mediated isothermal amplification assay (Takano et al., 2019). These tools could rapidly, accurately, sensitively, and specifically identify and characterize P. aeruginosa only with a simple an apparatus that could maintain a constant temperature (Wang et al., 2015), which demonstrated the potential to be applied in resource-limited or rural regions. However, the results identification and reporting systems of the isothermal amplification techniques normally rely on indicator, fluorescent dye or fluorescent probe, and amplification bias and non-specific amplification inherent in exponential strategies (Zhao et al., 2015). Therefore, the urgent need for new nucleic acid detection techniques with rapidity, accuracy, and high sensitivity still exists.

Discovery of the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein (Cas) (CRISPR-Cas) system has revolutionized the biosensing field and sparked great interest in nucleic acid detection technologies (Li et al., 2019). The CRISPR-Cas biosensing system could transfer the sequence information of targets to detectable signals (such as fluorescence or colorimetric values) by employing the collateral cleavage activities of the Cas effectors (Cas12a, Cas12b, Cas13, and Cas14), conferring this technology high sensitivity and specificity of detection and simplicity to develop, which also exhibits great potential in point-of-care tests (Gootenberg et al., 2018; Myhrvold et al., 2018; Bonini et al., 2021; Jirawannaporn et al., 2022; Kumaran et al., 2023; Zheng et al., 2023). In particular, by coupling isothermal amplification procedure, the detection performance of CRISPR-Cas biosensing system is greatly improved, and the target type also can be converted (Li et al., 2019; Zhang et al., 2023). Recently, the CRISPR-Cas–based biosensing detection platforms, such as SHERLOCK (Specific High Sensitivity Enzymatic Reporter Unlocking, RPA combination with Cas13a) (Myhrvold et al., 2018) and DETECTR (DNA Endonuclear Targeted Crispr Trans Reporter, RPA combination with Cas12a) (Chen et al., 2018), have been rapidly developed and already commercial available for pathogen detection.

In this study, a CRISPR-Cas12a–based RPA detection platform (CRISPR-RPA) targeting the oprL gene was developed and validated for rapid, accurate, sensitive, and specific diagnosis of P. aeruginosa infection. This two-step detection platform included oprL gene amplification using RPA assay at 42°C within 20 min and CRISPR-Cas12a detection at 37°C for 10 min. The result was interpreted using real-time fluorescence analysis using the single-strand DNA (ssDNA) reporter (5′-FAM-TTATTAT-BHQ1-3′). The detection performance of the P. aeruginosa–CRISPR–RPA assay was confirmed with DNA templates of P. aeruginosa strains, other respiratory pathogen strains, and clinical samples.

P. aeruginosa was one of the most common pathogens of hospital-acquired pneumonia (HAP) (16.9%–22.0%) (Moradali et al., 2017; Reynolds and Kollef, 2021) and also accounted for at least 1.0% of community-acquired pneumonia (Fine et al., 1996). Moreover, it was reported that 27.7% of the P. aeruginosa strains isolated from patient with HAP admitted in Intensive Care Unit (ICU) were of carbapenem resistance (Botelho et al., 2019). The high disease burden caused by P. aeruginosa and the increasing trend of antimicrobial resistance even multi-drug resistance of P. aeruginosa strains challenged the public health globally, and improvements to increase P. aeruginosa identification rate and time were urgently needed.

The CRISPR-Cas biosensing system has inspired numerous research activity in the diagnostic area on nucleic acid detection platform development (Li et al., 2019; Li et al., 2021; Jia et al., 2023), and the recently well-developed nucleic acid detection methods (such as SHERLOCK, HOLMES, DETECTR, and HUDSON) have manifested this. These methods were mainly developed with various CRISPR-Cas effectors (such as 12a, 12b, 13a, and 13b), which normally possess trans-cleavage activity, and activation of the trans-cleavage activity commonly required the formation of Cas effector/crRNA/target DNA ternary complex (Li et al., 2018a; Li et al., 2019). For example, the CRISPR-Cas12a effector could target DNA and trans-cleave any collateral ssDNA (Zetsche et al., 2015). Only after recognizing the target sequence that complementary to the crRNA sequence and juxtaposed with a suitable protospacer-adjacent motif (PAM, TTTN), the trans-cleavage activity of the CRISPR-Cas12a effector was able to be activated, following the paired fluorescence/quencher-labeled ssDNA probe cleaved and a fluorescent readout generated, which could be monitored by the real-time fluorescence detector (Li et al., 2018b). Owing to its merits of being highly efficient, sensitive, ultra-specific, and time-efficient, the CRISPR-Cas biosensing system has attracted much attention for its application in molecular diagnostic field. Therefore, in this study, we integrated the CRISPR-Cas12a biosensing system with RPA isothermal amplification techniques to optimize the P. aeruginosa identification rate and efficiency.

Compared with other nucleic acid amplification techniques, RPA was more preferable due to its simplicity, sensitivity, extremely rapidity, operation at low and constant temperature and with simple instruments, and no need for multiple primers (Lobato and O'Sullivan, 2018). In this study, the RPA pre-amplification step could be completed within 20 min at 42°C only with a simple water bath that could sustain a constant temperature. Because the reagents of RPA were freeze-dried and stored in the reaction tube, the RPA kit was especially convenient to store and employed. Thus, in this study, by combining with CRISPR-Cas12a detection platform, the P. aeruginosa–CRISPR–RPA assay was able to be performed independent of sophisticated equipment and foregoing the need for maintenance of a cold chain, which were attractive for use in point-of-care diagnostics and rural areas. Moreover, after optimization, the detection procedure of P. aeruginosa–CRISPR–RPA assay could be completed within half an hour, including 20 min for RPA reaction and 10 min for CRISPR-Cas12a detection, which was apparently rapid than that of PCR-based method and other isothermal amplification methods. In general, the P. aeruginosa–CRISPR–RPA assay in this study ensured its high sensitivity by pre-amplification the target nucleic acid using the attractive RPA method and guaranteed its high specificity with both the specific RPA primers and gRNA, together with paired fluorescence/quencher-labeled ssDNA probe, producing accurate and easy-to-interpret readouts.

The new established P. aeruginosa–CRISPR–RPA assay was proven sensitive to detect P. aeruginosa strains. It can detect as low as 60 fg (~8 copies) of P. aeruginosa genomic DNA per reaction, obviously more sensitive than the RPA-only method that detected by agarose gel electrophoresis method (6 pg). When compared with the previously reported P. aeruginosa–MCDA assay (100 fg) (Wang et al., 2020), the P. aeruginosa–CRISPR–RPA assay also exhibited higher sensitivity. However, the sensitivity of the P. aeruginosa–CRISPR–RPA assay was slight lower than the RPA-LFS assay developed by Yang et al. in 2021 (3.05 copies per reaction) (Yang et al., 2021). Thus, further optimization of the P. aeruginosa–CRISPR–RPA assay is still needed to improve the detection sensitivity, and more comparisons will be carried out to provide better reference to the clinicians for rapid and accurate diagnosis of P. aeruginosa–associated infections.

The specificity of the P. aeruginosa–CRISPR–RPA assay was evaluated by 17 non–P. aeruginosa strains, most of which were common respiratory pathogens. After detected by the P. aeruginosa–CRISPR–RPA assay, none of the 17 non–P. aeruginosa strains displayed a positive result except for the eight P. aeruginosa strains, manifesting that the P. aeruginosa–CRISPR–RPA assay was specific enough for diagnosis of P. aeruginosa infection. However, an obvious drawback of the specificity evaluation test was that no other members of genus Pseudomonas strains was tested here; thus, it will be supplemented if available in the future. After all, the P. aeruginosa–CRISPR–RPA assay was highly specific to detect P. aeruginosa strains and had no cross-reactivity with other pathogens.

Finally, the clinical validity of the P. aeruginosa–CRISPR–RPA assay was evaluated using 96 BALF samples initially diagnosed by MFC method. The P. aeruginosa–CRISPR–RPA assay reported 19 P. aeruginosa–positive samples and 77 negative samples, all of which was consisted with results by the MFC method, implying the P. aeruginosa–CRISPR–RPA assay was reliable for P. aeruginosa detection. Moreover, the P. aeruginosa–CRISPR–RPA assay was able to report the results of these clinical samples within half an hour, whereas that by MFC method needs about an hour, further demonstrating the superiority of the P. aeruginosa–CRISPR–RPA assay. It was well-known that conventional culture–based technique was more proper to be employed to validate the new established P. aeruginosa–CRISPR–RPA assay; however, no original clinical BALF samples was available; thus, comparison of the performance of this new method with culture-based technique could only be carried out in the future studies. Together, it could be concluded that the validity was a promising tool for the rapid and accurate diagnosis of P. aeruginosa infection.

Certainly, there are still some limitations in this study: (1) the genetic information of the eight P. aeruginosa strains was not available, which may affect the evaluation of the new established detection system in correctly P. aeruginosa identification; (2) the background fluorescence signal is occasional high, which may lead to false-positive results; (3) the carryover contamination cause by opening the RPA amplification tube may produce background signals; and (4) more clinical samples should be tested for clinical validation. Of course, there are also some improvements that can be made in the future, including using more genetically diverse strains for the method establishment and verification, interpreting the result of P. aeruginosa–CRISPR–RPA assay under blue light if the drawback of high background signals solved; moreover, conducting the whole detection procedure within one step if further optimizations were provided, which also can avoid the production of aerosol pollution.

In summary, we reported the development and validation of a CRISPR-Cas12a–based detection platform for P. aeruginosa identification and termed it P. aeruginosa–CRISPR–RPA assay. The two-step P. aeruginosa–CRISPR-RPA assay was capable of detecting P. aeruginosa only within half an hour with simple instruments. After detecting the serial dilutions of P. aeruginosa genomic DNA, other non–P. aeruginosa strains and clinical samples with the P. aeruginosa–CRISPR-RPA assay, it can be concluded that the P. aeruginosa–CRISPR–RPA assay possesses the merits of rapidity, reliability, easy to perform, higher sensitivity, and specificity. Thus, the P. aeruginosa–CRISPR–RPA assay established here was a reliable and promising tool for early and rapid diagnosis of P. aeruginosa infection and stop of its wide spread especially in the hospital settings.

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 ethic committee of Capital Institute of Pediatrics. The studies were conducted in accordance with the local legislation and institutional requirements. The human samples used in this study were acquired from primarily isolated as part of your previous study for which ethical approval was obtained. Written informed consent for participation was not required from the participants or the participants’ legal guardians/next of kin in accordance with the national legislation and institutional requirements.

SL performed the experiments, analyzed the data, and drafted the manuscript. SH, FL, and YS helped perform the experiments and organize the data. JF, FX, NJ, XH, and CS provided experimental reagents and materials. JZ and YW supervised this study and revised the manuscript. DQ conceived, supervised and funded this study, and revised manuscript. All authors contributed to the article and approved the submitted version.