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Development of RPA-Cas12a assay for rapid and sensitive detection of Pneumocystis jirovecii

BMC Microbiology volume 24, Article number: 314 (2024) Cite this article

Abstract

Pneumocystis jirovecii is a prevalent opportunistic fungal pathogen that can lead to life-threatening Pneumocystis pneumonia in immunocompromised individuals. Given that timely and accurate diagnosis is essential for initiating prompt treatment and enhancing patient outcomes, it is vital to develop a rapid, simple, and sensitive method for P. jirovecii detection. Herein, we exploited a novel detection method for P. jirovecii by combining recombinase polymerase amplification (RPA) of nucleic acids isothermal amplification and the trans cleavage activity of Cas12a. The factors influencing the efficiency of RPA and Cas12a-mediated trans cleavage reaction, such as RPA primer, crRNA, the ratio of crRNA to Cas12a and ssDNA reporter concentration, were optimized. Our RPA-Cas12a-based fluorescent assay can be completed within  30–40 min, comprising a 25–30 min RPA reaction and a 5–10 min trans cleavage reaction. It can achieve a lower detection threshold of 0.5 copies/µL of target DNA with high specificity. Moreover, our RPA-Cas12a-based fluorescent method was examined using 30 artificial samples and demonstrated high accuracy with a diagnostic accuracy of 93.33%. In conclusion, a novel, rapid, sensitive, and cost-effective RPA-Cas12a-based detection method was developed and demonstrates significant potential for on-site detection of P. jirovecii in resource-limited settings.

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Introduction

Pneumocystis jiroveci (P. jiroveci) remains a significant cause of pneumonia in immunocompromised patients [1, 2]. Although infections with this pathogen have decreased with the widespread use of highly active antiretroviral therapy for human immunodeficiency virus infection, these infections continue to pose a substantial threat to individuals with weakened immune systems, particularly those with acquired immunodeficiency syndrome and other immunocompromised conditions [3]. The recent study evaluating pediatric pneumonia through molecular diagnostics in the developing world found pneumocystis pneumonia (PCP), caused by P. jiroveci, accounted for up to 1–2% of cases of community-acquired pneumonia among children under 5 years old, with a peak incidence in infants [4].

Due to the challenges of culturing P. jiroveci in vitro, the diagnosis traditionally depends on clinical symptoms, radiographic findings, and visual examination of the clinical specimen, such as bronchoalveolar lavage fluid or induced sputum, using the staining methods [5,6,7]. However, diagnosing PCP clinically presents a considerable challenge due to its nonspecific symptoms, inconclusive imaging characteristics, and co-infection with various pathogens such as cytomegalovirus [8]. These complex conditions necessitate early, prompt and accurate diagnosis for timely treatment and improved outcomes. In recent decades, molecular methods, including polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP), and antibody-antigen testing techniques on less invasive specimen sources, have been exploited for the detection of P. jirovecii [9,10,11]. Among these, PCR has emerged as a pivotal diagnostic tool for P. jirovecii due to its high sensitivity [12,13,14]. PCR is estimated to make up 86–100% of analyses conducted on bronchoalveolar lavage fluid (BALF), aspirates, and induced sputum specimens [15]. Nevertheless, the need for the costly equipment makes PCR an impractical choice in resource-limited settings [16]. In contrast to the PCR method, LAMP, which operates at relatively low temperatures, has facilitated accessible and user-friendly diagnosis of P. jirovecii in resource-constrained environments [11]. A problematic shortcoming of this method is the high risk of contamination, which often leads to false positives in negative controls [17].

Another novel molecular diagnostic technology, combining isothermal amplification, recombinase polymerase amplification (RPA), and CRISPR-Cas12a, has garnered significant attention for its user-friendly nature and high accuracy [18]. This combination offers advantages such as simple operation, rapid amplification speed, and reactions occurring at 37–42 °C, among others. Furthermore, the CRISPR-Cas12a-mediated detection reaction occurring at 37–39 °C demonstrates the advantages of signal amplification, high sensitivity, and high specificity in nucleic acid detection [19]. The biosensor analytical technology based on CRISPR-Cas12a has been exploited for great potential in the rapid detection of infectious pathogens. Therefore, this combination has been extensively developed for the detection of various pathogens in diverse scenarios [20, 21]. To date, there have been no reports on the detection of P. jirovecii using the combination of RPA and CRISPR-Cas12a.

In this study, we introduced a rapid and sensitive RPA-Cas12a-based assay for detecting P. jirovecii by targeting a conserved gene sequence. The developed method can detect target DNA at a concentration of 0.5 copies/µL within 35–45 min, making it easily applicable in resource-limited settings.

Materials and methods

Artificial samples

200 mL of artificial BALF purchased from Biochemazone™ were used. Among these, each 5 mL of artificial BALF was supplemented with cell culture from A549 cells at a final concentration of 106 cells/mL. These were then mixed with serially diluted solutions of the positive plasmid containing the conserved target sequence of P. jirovecii, resulting in 18 artificial BALF samples ranging from 1.0 × 102 copies/mL to 1.0 × 106 copies/mL. Additionally, 12 artificial BALF samples were used without any additional components as the negative control. The artificial BALF samples were extracted using TaKaRa MiniBEST Universal Genomic DNA Extraction Kit Ver.5.0 by TaKaRa Co., Ltd. (Dalian, China) following the manufacturer’s instructions. The extracted genome was used as template DNA for the RPA and qPCR assay, respectively.

Reagents

The RPA kit was obtained from Jiangsu Lesun Biotechnology Co., Ltd (Wuxi, China), while Cas12a and its reaction buffer NEB buffer 2.1 were acquired from New England Biolabs (MA, USA). The ssDNA FQ reporter was synthesized by GENEWIZ Inc. (Suzhou, China) and labeled with 6-FAM and BHQ1 at its 5′ and 3′ ends.

Preparation of standard DNA of the target region of P. jirovecii

Twenty complete genome sequences of P. jirovecii, including U07226.1, JQ365746.1, AB481404.1, AB469817.1, MF988682.1, MW646091.1, MK300654.1, OR475700.1, KC470771.1, MT780540.1, OR475687.1, KC470809.1, MF989107.1, MF989234.1, JF442099.1, MW646090.1, MK120007.1, MF980974.1, MF989232.1, and FJ164067.1, were obtained from the National Center for Biotechnology Information and aligned for a comparative analysis of genomic sequences using MEGA X. The analysis revealed that the gene segment of the 5.8 S ribosomal RNA is highly conserved and was identified as the target region (Fig. S1). Subsequently, the conserved fragment, comprising approximately 488 nucleotides, was synthesized by Shanghai Sangon biotech (Shanghai, China), and was then cloned into the pUC-57 to generate pUC57-PNC. The resulting recombinant plasmid was stored at -80 °C for further use. The copy number of standard DNA for the P. jirovecii target region was determined using a previously reported method [22].

In vitro preparation of RPA primers and crRNA

Seven RPA primers and four crRNAs were designed based on the conserved sequence for P. jirovecii, and the corresponding oligonucleotide sequences were synthesized by Shanghai Sangon Biotech Co., Ltd (Table 1). RPA primers were designed using IDT OligoAnalyzer based on the conserved sequence of the target sequence. For crRNA design in CRISPR Cas12a-based assays, the CRISPR Design Tool was utilized. The secondary structure and dimer analysis of RPA primer were performed using Oligo Analyzer 3.1. The preparation of crRNA involved a two-step process following a previously reported method [21]. Initially, four pairs of oligonucleotides were annealed to produce double-stranded DNAs (dsDNA), which were then transcribed using the IVT T7 Kit. The IVT reaction mixture, comprising 5 µL of 10× transcription buffer, 5 µL of each NTP solution (20 mM), 1.25 µL of RNase inhibitor (2 U/µL), 5 µL of T7 RNA polymerase (5 U/µL), 16.25 µL of RNase-free water, and 2.5 µL of annealed dsDNA (100 ng/µL), was incubated at 42 °C for 6 h. Subsequently, the crRNA products underwent phenol/chloroform extraction, and the concentration of crRNA was determined using a spectrophotometer (Metash Instruments, Shanghai, China).

Table 1 The oligonucleotide sequences for the primer, crRNA, and ssDNA reporter
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Optimization of reaction parameters for RPA and Cas12a-mediated assay

The RPA-Cas12a-mediated assay is divided into two steps. The first step involves the exponential amplification of the target sequence by RPA. A 25 µL RPA reaction mixture was incubated in the Axxin T8 isothermal instrument at 38 °C for 20–40 min. The mixture comprised 12.5 µL of reaction buffer, 2 µL of each primer (10 µM), 6.5 µL of ddH2O, 2.5 µL of standard DNA (104 copies/µL), and 1.5 µL of 280 mM magnesium acetate. DNase-free water served as the template for the negative control in the same volume. To determine the optimal RPA primer combination and concentration ranging from 200 nM to 800 nM, reaction time (20 min, 25 min, 30 min, 35 min and 40 min), as well as the reaction temperature (37 °C, 38 °C, 39 °C, 40 °C, and 41 °C), the amplification efficiencies of RPA were evaluated through agarose gel electrophoresis and/or real-time fluorescence signal detection analysis. After identifying the optimal primer combination, further analysis was conducted with different primer concentrations to determine the optimal reaction concentration for the primer combination.

In the second stage, the Cas12a-mediated trans-cleavage reaction was conducted in a 25 µL reaction mixture containing 2.5 µL of 10 × NEB Buffer 2.1, 4 µL of ssDNA reporter (1 µM), 1.5 µL of 1 µM Cas12a, 4.5 µL of 1 µM crRNA, 7.5 µL of deionized water, and 5 µL of RPA products or deionized water (as a negative control). The Cas12a-mediated reaction was performed at 37 °C for 30 min. Fluorescent signals were recorded every 10 s using ssDNA reporter as the substrate to yield the assay results. Alternatively, after the reaction was complete, the reaction tube was placed under a UV lamp for visual inspection to check if the reaction mixture turned green. At this stage, the optimal crRNA and the optimal ratio of crRNA to Cas12a and ssDNA, the trans-cleavage efficiencies of four crRNAs were assessed using the Cas12a complex in combination with the target sequence. The evaluation of trans-cleavage efficiencies was based on the intensity of the fluorescent signal. Furthermore, the optimal ssDNA reporter concentration was determined using the intensity of the fluorescent signal and naked-eye observation under UV exposure.

Specificity and sensitivity of the RPA-Cas12a-mediated assay

To evaluate the specificity of the RPA-Cas12a-mediated assay, standard DNA of the target region of P. jirovecii and various fungal genomic DNA samples were used as templates. These samples included Canidia albicans (C. albicans), Crytococcus neofonmans (C. neoformans), Candida parapsilosis (C. parapsilosis), Candida guilliermondii (C. guilliermondii), Candida tropicalis (C. tropicalis), and Candida glabrata (C. glabrata). The template amount per reaction was standardized to a final concentration of 105 copies/µL. Additionally, a concentration gradient of standard DNA of the target region (10000, 1000, 100, 10, 1, 0.5, 0.1, and 0 copies/µL) was used as the template to examine the sensitivity of the RPA-Cas12a-mediated assay. The results were analyzed using real-time fluorescence readout, with each reaction replicated three times.

qPCR detection

To evaluate the performance of the RPA-Cas12a-mediated assay, nucleic acids from artificial samples were assessed and compared using both the RPA-Cas12a-mediated assay and qPCR. The total DNAs from 30 artificial samples in this study were extracted and utilized as templates. The reaction was conducted in a 20 µL reaction mixture, comprising 10 µL of 2 × qPCR buffer, 0.8 µL of enzyme, 0.4 µL of each primer (10 µM), 0.4 µL of ROX Dye II (10 µM), 2µL of template, and 6.4 µL of DNase-free ddH2O. The qPCR assay was performed using the P. jirovecii kit (GeneoDx Biotech Co., Ltd., Shanghai, China) in an ABI 7500 (Applied Biosystems) instrument. The reactions included an initial step of pre-denaturation at  95 °C for  1 min, followed by 40 cycles of 95 °C for 30 s and 58 °C for 30 s.

Statistics

The data presented are representative of each sample with a minimum of three independent biological replicates. Statistical analysis of fluorescence values was conducted using Origin (Originlab Software, version 2023b), with statistical differences calculated using the Student’s t-test. The agreement between the two methods was assessed using Cohen’s“kappa” (κ) analysis via the Diagnostic Test using openepi software.

Results

Optimization of the primer pair and its concentration for RPA assay

To establish a basic detection system for P. jirovecii DNA for use in optimizing RPA reaction conditions, various combinations of primer pairs and crRNA for the RPA-Cas12a-mediated assay were optimized in this study. Seven primer combinations were evaluated through RPA assays using a final concentration of 10,000 copies/µL of standard DNA of the target region as the template. Subsequently, the RPA products generated by the different primer pair combinations were assessed based on intensity (none, faint, or clear). The results, illustrated in Fig. 1, clearly showed that the F2/R1 combination exhibited the highest sensitivity and produced a distinct 246 bp DNA band, consistent with the expected outcome. Furthermore, the F2/R1 concentration was optimized, revealing a positive relationship between the final primer concentration ranging from 200 nM to 300 nM and the RPA amplification efficiency. Conversely, an inverse correlation with the RPA amplification efficiency was observed for final concentrations ranging from 300 nM to 800 nM (Fig. 2A). Similarly, the amplification efficiency was positively associated with reaction time, reaching saturation by 30 min (Fig. 2B). Evaluation of the reaction products at 37, 38, 39, 40, and 41 °C based on fluorescence intensity showed excellent efficiency at temperatures of 37, 38, 39, and 40 °C, with slightly lower efficiency at 41 °C (Fig. 2C). Considering the portability of the reaction conditions, 38 °C was selected for subsequent experiments. Consequently, the primer combination F2/R1, with a final concentration of 300 nM, a reaction temperature of 38 °C, and a reaction time of 30 min, was chosen for further RPA experiments.

Fig. 1
figure 1

Screening of the optimal primer combination for RPA was conducted. and the amplification products of RPA were analyzed using agarose gel electrophoresis (A) and fluorescence intensity measurements (B). A, 1 for the combination of F1 and R1; 2, for the combination of F1 and R2; 3, for the combination of F2 and R1; 4, for the combination of F2 and R2; 5, for the combination of F3 and R3; 6, for the combination of F4 and R3; 7, for the combination of F1, F2 and R1, R2; 8, Negative control

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Fig. 2
figure 2

The effect of RPA primer concentration (A), RPA reaction time (B), and RPA reaction temperature (C) on RPA amplification efficiency using the CRISPR-Cas12a detection system

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Optimizing parameters that affect Cas12a-mediated trans cleavage activity

First, the effectiveness of four crRNAs candidate and the ratio of crRNA were tested to achieve optimal trans-cleavage activity, a critical parameter for the Cas12-mediated method. In this experiment, the final concentrations of each crRNA and Cas12a were standardized at 80 nM and 60 nM, respectively. As shown in Fig. 3A, the findings demonstrated that crRNA1 and crRNA2 generated detectable fluorescence signals through the formation of a ternary complex with Cas12a and the target sequence. Remarkably, crRNA1 exhibited the strongest trans-cleavage activity with a prominent fluorescence signal, achieving a reaction plateau within the following 25 min. In contrast, crRNA3 and crRNA4 showed virtually no detection signal, indicating their relatively poor performance in the assay.

Fig. 3
figure 3

Evaluation of the optimal crRNA and the optimal ratio of the crRNA to Cas12a in the RPA-Cas12a-based fluorescence assay for P. carinii detection. The fluorescence readouts of the RPA-Cas12a real-time assay were recorded following incubation with various crRNAs (A) and different ratios of the optimal crRNA to Cas12a (B) for 30 min at 37 °C. (C-F) Predicted structure of four crRNAs plotted using the SnapGene software

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Furthermore, the ratio of crRNA1 to Cas12a protein was optimized, with the Cas12a protein concentration fixed at 60 nM. The results exhibited that the optimal fluorescence signal was achieved when the ratio of crRNA1 to Cas12a protein was 4:1, peaking within 10 min (Fig. 3B). Consequently, crRNA1, a Cas12a protein concentration of 60 nM, and a 2:1 ratio of crRNA1 to Cas12a protein were selected for the subsequent Cas12a-mediated detection assay. To explore the underlying reasons, the structures of the four crRNAs were analyzed. As shown in Fig. 3C-F, in the crRNA1 construct, the sequence positions 20–22, complementary to positions 8–10, enhanced Cas12a cleavage efficiency, possibly due to the specific folding of the tail onto the spacer. Additionally, the spacer sequence of crRNA1 exhibited no intra-array complementarity, indicating that the crRNA structure may significantly influence its efficiency.

Next, various concentrations of the ssDNA reporter were investigated. As shown in Fig. 4A, a strong fluorescence signal was detected with increasing concentrations of the ssDNA reporter up to 240 nM, indicating an enhanced trans-cleavage activity. Additionally, within the ssDNA concentration range of 200–240 nM, a visible green color change could be directly observed by the naked eye under UV light (M-15E UV Transilluminator, USA) in the reaction system (Fig. 4B). Therefore, ssDNA reporter concentrations of 240 nM were chosen for Cas12a-mediated fluorescence and naked-eye observation detection.

Fig. 4
figure 4

Optimization of the ssDNA concentration for Cas12a was performed. The readouts of the RPA-Cas12a assay, utilizing real-time fluorescence values (A) and visual observation (B), were assessed after incubation with different ssDNA concentrations for 30 min at 37 °C

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Specificity of the RPA-Cas12a-mediated fluorescence assay with the optimized reaction condition

Various samples, including standard DNA of target sequences, genomic DNA of C. albicans, C. neoformans, C. parapsilosis, C. guilliermondii, C. tropicalis and C. glabrata, were used to assess the specificity of RPA-Cas12a-mediated assay. As shown in Fig. 5A, in the RPA-Cas12a-mediated real-time fluorescence assay, only the standard DNA of the target sequence yielded detectable fluorescence signals, unlike the other samples. This observation indicated that no cross-reactivity was observed for other fungal samples. Moreover, the quantitative data from the RPA-Cas12a-mediated fluorescence assay were determined based on the end-point fluorescence values. As shown in Fig. 5B, a significant difference in fluorescence values was evident only between the group with the addition of standard DNA and the negative group (p < 0.001). These results suggested robust specificity in the RPA-Cas12a-mediated fluorescence assay for detecting P. jirovecii.

Fig. 5
figure 5

Specificity testing of the RPA-Cas12a-based fluorescence assay for P. jirovecii detection using the standard DNA of P. carinii and genomic DNA extracted from Canidia albicans (C. albicans), Candida parapsilosis (C. parapsilosis), Candida guilliermondii (C. guilliermondii), Candida tropicalis (C. tropicalis), and Candida glabrata (C. glabrata) as templates. Only the standard DNA of P. carinii exhibited fluorescence signals in both real-time (A) and end-point (B) fluorescence readings. The sensitivity of the RPA-Cas12a-based fluorescence assay was assessed by observing an increase in fluorescence intensity with rising concentrations of the target gene template (ranging from 0 to 10,000 copies/µL) in both real-time (C) and end-point (D) fluorescence measurements. Standard deviations of the data (n = 3) are represented by error bars. Statistical analysis was conducted to compare the various concentration groups with the negative control (NC) at the corresponding time points. Significance levels were indicated as ** for p < 0.05, *** for p < 0.001, and n.s. for non-significant differences

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Sensitivity of the RPA-Cas12a-mediated fluorescence assay with the optimized reaction condition

A series of concentrations of target standard DNA, ranging from 0, 0.1, 0.5, 1, 10, 100, 1000 to 10,000 copies/µL, was employed to assess the sensitivity of the RPA-Cas12a-mediated assay. As shown in Fig. 5C, fluorescence values increased with the rising concentrations of target standard DNA, and a final concentration of the target standard DNA equal to or greater than 0.5 copies/µL yielded a clear fluorescent signal. Additionally, a significant difference in fluorescence values was observed between a target standard DNA concentration of 1 copies/µL and the negative control after a 20-minute reaction (Fig. 5D). These findings indicated that a high sensitivity of 12.5 copies of target DNA per reaction was achieved in the RPA-Cas12a-mediated fluorescence assay for detecting P. jirovecii.

Examination of the performance of the RPA-Cas12a-mediated fluorescence assay

To examine the performance of RPA-Cas12a-mediated fluorescence assay, the extracted DNA samples from 30 artificial samples were simultaneously tested using both the RPA-Cas12a-mediated fluorescence assay and qPCR. A diagnostic accuracy of 93.33% was achieved between the RPA-Cas12a-mediated fluorescence assay and the qPCR assay for P. jirovecii-artificial samples (Table 2). Cohen’s “kappa” (κ) analysis demonstrated a strong agreement between the two methods, yielding aκ-value of 0.8571 with a p-value of 0.06667. These results exhibit a robust diagnostic agreement between the developed RPA-Cas12a-mediated fluorescence assay and qPCR.

Table 2 Statistical analysis of simulated samples using both the RPA-Cas12a-mediated assay and qPCR
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Discussion

P. jirovecii is a common opportunistic fungal pathogen that poses a significant risk of life-threatening PCP in immunocompromised individuals. Early and precise diagnosis is crucial for timely treatment initiation and improved patient outcomes [23, 24]. Traditional diagnostic methods, relying on staining and direct observation of the organisms in bronchoalveolar lavage fluid, are invasive and exhibit limited sensitivity. To address these challenges, molecular detection techniques such as PCR, LAMP, and antibody-antigen assays have emerged, offering noninvasive sample testing with heightened sensitivity. While qPCR is commonly used for P. jirovecii detection [25], its limitations in resource-limited settings have prompted the need for rapid, user-friendly, and cost-effective alternatives, such as isothermal nucleic acid amplification technology.

In this study, we developed an RPA-Cas12a-mediated fluorescence assay for P. jirovecii detection by identifying the target sequence within the 5.8 S ribosomal RNA gene region. Optimization of reaction parameters and primer design led to the selection of the optimal primer combination for RPA amplification. Additionally, the optimization of crRNA sequence design and the ratio of crRNA to Cas12a enhanced Cas12a-mediated trans-cleavage activity, resulting in efficient detection. The dual targeting strategy of RPA amplification and Cas12a-mediated trans cleavage improved specificity and efficiency while reducing off-target noise, achieving a low detection threshold of 12.5 copies of target DNA per reaction. Several recent studies have highlighted the rapid detection of P. jirovecii through LAMP [26], as well as employing a CRISPR/Cas13a-based assay [27]. Nevertheless, in the assay of LAMP, the detection threshold was determined to be approximately 4 × 103 copies/mL. Additionally, the CRISPR/Cas13a-based assay targets RNA for P. jirovecii detection, which is more susceptible to degradation than DNA, requiring heightened technical expertise during handling.

Subsequently, the conditions for the Cas12a-mediated trans cleavage reaction were fine-tuned. Despite the lack of definitive guidelines for optimal crRNA sequence design, the assay required experimental analysis of multiple crRNAs to determine cleavage activity. We initially focused on crRNA optimization, noting distinct fluorescence signals corresponding to nucleotide sequences of crRNA. Interestingly, crRNA1 exhibited the highest fluorescence signal when the handle sequence was extended, a result supported by a previous study indicating that the hairpin structure of the repeat and the avoidance of intra-array complementarity enhance Cas12a protein binding [28]. Structural analysis of crRNA1 suggested that these features could enhance Cas12a protein trans cleavage activity. Moreover, in addition to optimal structural binding with crRNA1, the trans cleavage activity mediated by Cas12a was influenced by the optimal crRNA to Cas12a ratio. Consequently, distinct fluorescence signals confirmed the activation of Cas12a trans cleavage activity based on the crRNA1 to Cas12a ratio.

Furthermore, in comparison to PCR and LAMP, the dual targeting strategy involving RPA amplification and Cas12a-mediated trans cleavage activity could enhance the specificity and efficiency of P. jirovecii detection while reducing off-target background noise. Notably, the developed RPA-Cas12a-mediated fluorescence assay achieved a lower detection threshold of 12.5 copies of target DNA per reaction (0.5 copies/µL). To evaluate the applicability of RPA-Cas12a-mediated fluorescence assay for clinical specimens, 30 artificial specimens were examined. The RPA-Cas12a-mediated assay yielded highly consistent results with the qPCR method, achieving a 93.33% diagnostic accuracy for P. jirovecii.

In conclusion, the RPA method, in combination with Cas12a, has been developed and could serve as a reliable tool for the detection of P. jirovecii. Our method has great potential for diagnosing P. jirovecii in various situations due to its convenience and speed.

Data availability

The datasets analysed during the current study are available in the [NCBI] repository, [U07226.1, JQ365746.1, AB481404.1, AB469817.1, MF988682.1, MW646091.1, MK300654.1, OR475700.1, KC470771.1, MT780540.1, OR475687.1, KC470809.1, MF989107.1, MF989234.1, JF442099.1, MW646090.1, MK120007.1, MF980974.1, MF989232.1, and FJ164067.1].

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Acknowledgements

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Funding

This work was supported by Ningbo Key Research and Development Project: Leading the Charge with Open Competition (2023Z200), Ningbo Key Laboratory of Virus Research (20221CXJD030031), Ningbo Top Medical and Health Research Program (2023020713), and the Shaanxi Province Scientist and Engineer Team Building Project (2024QCY-KXJ-063).

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Author notes

  1. Qiming Liu and Hao Zeng contributed equally to this work.

Authors and Affiliations

  1. School of Biological and Pharmaceutical Sciences, Shaanxi University of Science and Technology, Xi’an, 710021, China

    Qiming Liu, Hao Zeng, Ting Wang & Weidong Qian

  2. Ningbo Municipal Key Laboratory of Virology, Ningbo Municipal Center for Disease Control and Prevention, Ningbo, 315010, China

    HongXia Ni & Yongdong Li

  3. Department of Epidemiology and Biostatistics, School of Public Health Health Science Center of Ningbo University, Ningbo, 315211, China

    Ting Fang & Guozhang Xu

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  1. Qiming Liu
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  4. HongXia Ni
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  6. Weidong Qian
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Contributions

T. F., G. X. and W. Q. were responsible for conceptualizing the research. Q. L., T. W., and H. Z. devised the methodology. H. N. and Y. L. carried out the formal analysis and investigation. The original draft of the manuscript was prepared by H. Z. and W. Q. Subsequently, the writing process underwent review and editing by W. Q., T. W., and H. Z. G. X. and H. N. secured the funding for this project. T. F. supervised the overall study. Each author participated in revising the manuscript and lent their approval to the final version.

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Correspondence to Weidong Qian, Ting Fang or Guozhang Xu.

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Liu, Q., Zeng, H., Wang, T. et al. Development of RPA-Cas12a assay for rapid and sensitive detection of Pneumocystis jirovecii. BMC Microbiol 24, 314 (2024). https://doi.org/10.1186/s12866-024-03440-z

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Keywords

BMC Microbiology

ISSN: 1471-2180

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