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. 2026 Jul 4;98(7):e71032. doi: 10.1002/jmv.71032

A One‐Pot CRISPR‐Cas12b Assay for Rapid Detection of Human Adenovirus Serotypes 3 and 7

Linglong Wan 1, Juan Zhou 1, Lei Yu 2,3, Xiaolan Huang 1, Jin Fu 1, Fei Xiao 1, Nan Jia 1, Yu Zhang 1, Min Chen 1, Zhaomin Feng 4,5,✉, Yi Wang 1,6,7,8,✉
PMCID: PMC13349421  PMID: 42400407

ABSTRACT

Human adenovirus (HAdV) is a leading cause of acute respiratory tract infections (ARTIs) in children. The high prevalence of HAdV serotypes 3 and 7 in regions such as China presents a significant public health challenge. Here, we propose a one‐pot assay that integrates multiple cross displacement amplification (MCDA) with CRISPR‐Cas12b for the detection of HAdV‐3 and HAdV‐7, termed HAdV‐MCDA‐One. In this system, MCDA provides exponential target amplification, while the collateral cleavage activity of Cas12b enables secondary signal amplification. The entire reaction is performed isothermally at 60°C in a single tube, providing a fluorescent readout within 50 min, making the assay suitable for point‐of‐care testing (POCT). Leveraging the single‐base recognition capability of CRISPR‐Cas12b, the assay demonstrates high specificity, with no cross‐reactivity observed against the other 13 identified pathogens. The limit of detection was determined to be 1.59 copies per reaction using target plasmids. Moreover, when evaluated with 96 clinical pharyngeal swabs, the assay showed 100% concordance with quantitative PCR (qPCR), confirming its clinical reliability. These results demonstrate HAdV‐MCDA‐One as a rapid and robust tool for HAdV‐3 and HAdV‐7 detection, with significant potential for clinical diagnosis and public health surveillance.

Keywords: Cas12b, CRISPR, gRNA, human adenoviruses, multiple cross displacement amplification

1. Introduction

Human adenovirus (HAdV) is a non‐enveloped, double‐stranded DNA virus belonging to the family Adenoviridae [1]. Based on genomic organization and biological characteristics, HAdV is categorized into seven species (A–G) [2]. HAdV exhibits broad tissue tropism, causing infections across multiple anatomical sites, including the respiratory, ocular, gastrointestinal, and urinary tracts [3]. As a leading etiological pathogen of acute respiratory tract infections (ARTIs) in pediatric patients, HAdV‐3 and HAdV‐7 are the dominant circulating serotypes in China [4]. While most infections are mild, immunocompromised individuals are at high risk of developing severe pneumonia, which is associated with significant mortality [3, 5, 6, 7]. Furthermore, HAdV's environmental resistance and high transmissibility contribute to localized outbreaks in crowded settings such as schools, military facilities, and kindergartens [8]. This underscores a pressing need for accurate and rapid detection methods for HAdV‐3 and HAdV‐7 to mitigate transmission and improve public health outcomes.

Currently, various diagnostic strategies are employed for HAdV identification, including virus culture, serological testing, and nucleic acid detection. Despite substantial advances, several drawbacks constrain their widespread application. Virus culture is limited by a prolonged turnaround time, rendering it unsuitable for early diagnosis [9]. Serological testing can be affected by cross‐reactivity due to conserved antigenic epitopes among different HAdV serotypes [10]. While PCR‐based molecular diagnostics are highly sensitive, their application is often hindered by a dependency on PCR cycler, complex protocols, and high cost [11].

The advent of isothermal amplification technology (IAT) has revolutionized nucleic acid detection. IAT platforms, such as loop‐mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA), enable rapid nucleic acid amplification at a constant temperature [12, 13, 14]. This feature eliminates the need for thermal cyclers, making them ideal for point‐of‐care testing (POCT) in primary laboratories or field settings. Multiple cross displacement amplification (MCDA), a recently developed IAT, exhibits great potential for the rapid and sensitive detection of target sequences and has been applied to diagnose various pathogens [15, 16, 17]. This method achieves nucleic acid amplification at a steady temperature (typically 58°C–69°C) within 40 min using only a basic isothermal instrument.

The occurrence of the trans‐cleavage activity of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and associated (Cas) proteins sparked great interest in biotechnology, particularly for nucleic acid detection [18, 19, 20]. Previous theories have expressed the synergistic potential of combining CRISPR‐Cas systems with IAT to achieve high sensitivity [21, 22]. In such systems, CRISPR‐Cas amplifies the signal following target amplification by IAT, reducing false positives that can arise from non‐specific products [23]. However, many existing protocols involve complex, multi‐step procedures that increase the risk of contamination. Therefore, developing simpler and more integrated CRISPR‐based detection platforms remains a key objective.

In this research, we invented a one‐pot CRISPR‐based method for the testing of HAdV, which we term HAdV‐MCDA‐One. This platform integrates MCDA amplification and CRISPR‐Cas12b detection within a single tube for the reaction free from the need to transfer liquids. Our evaluation demonstrates that the HAdV‐MCDA‐One platform is a robust method for diagnosing HAdV‐3 and HAdV‐7 infection, offering significant advantages for POCT and field surveillance applications.

2. Materials and Methods

2.1. Primer and gRNA Design

Hexon gene (RefSeq AC_000018, 18666‐21470) of HAdV, a relatively conserved gene, was chosen as the target gene to design primers and gRNA. Based on the mechanism of MCDA and the CRISPR/Cas12b system, we generated a set of 10 primers, gRNA, and ssDNA probes. The primer specificity was inspected by Primer‐Blast, and the sequences were aligned with those of other species by MEGA 11 and ESPript 3.0 for further verification (Figure S1). All these sequences and modifications are shown in Table S1.

2.2. Reagents and Apparatus

The EasyPure Viral DNA/RNA Kit and EasyPure Genomic DNA Kit for nucleic acid extraction and purification were purchased from TransGen Biotech Co. Ltd. (Beijing, China). The DNA Isothermal® Amplification Kit (containing 2× buffer) and AapCas12b (containing 10× buffer) were purchased from HuiDeXin Biotech Co. Ltd. (Tianjin, China). MCDA primers and ssDNA probe were procured from SinoGenoMax Co. Ltd. (Beijing, China). gRNA is provided by GenScript Co. Ltd. (Nanjing, China). The real‐time turbidimeter (LA‐320C) was supplied by Eiken Chemical Co. Ltd. (Japan). The ABI 7500 real‐time fluorescent quantitative PCR system was purchased from Applied Biosystems (USA).

2.3. Preparation of Plasmid and Clinical Sample

We cloned hexon gene (RefSeq AC_000018, 18666‐21470) for HAdV‐3 and HAdV‐7, a relatively conserved sequence, and constructed recombinant plasmids with pUC57. The standard plasmids serve as templates for subsequent validation. Additionally, the Chinese Center for Disease Control and Prevention (CDC) provided all non‐HAdV pathogens for specificity analysis, and the genomic DNA was extracted using EasyPure Genomic DNA Kit. Moreover, we obtained 96 clinical samples from the pharyngeal swab samples of outpatients provided by the Capital Centre for Children's Health, Capital Medical University. All these nucleic acids of clinical samples were acquired via the EasyPure Viral DNA/RNA Kit and preserved at −20°C until utilization.

2.4. Standard MCDA‐One Reaction

The standard reaction system was as previously reported [24]. In brief, 12.5 μL 2✕ isothermal reaction buffer, 0.4 μM each of displacement primers (F1 and F2), 0.8 μM each of amplification primers (C1, C2, D1, D2, R1, and R2), 0.4 μM each of cross primers (CP1* and CP2), 8 U Bst 2.0 DNA polymerase, 4 μL complex (150 nM gRNA and 100 nM AapCas12b incubated at 37°C for 10 min), 50 μM ssDNA probe, and 1 μL template were gathered in one tube. The volume was adjusted to a final volume of 25 μL with distilled water. This amplification system was incubated at 60°C for 50 min (initial amplification 10 min and 40 cycles for fluorescence readout). It is worth mentioning that the complex of gRNA and AapCas12b should be used immediately after preparation or stored at 4°C for 12 h before use.

2.5. Optimization of HAdV‐MCDA‐One

Reaction temperature for MCDA reaction was evaluated from 58°C to 65°C with an interval of 1°C by a real‐time turbidimeter to obtain the optimal one. Considering the activity of the AapCas12b enzyme, temperatures spanning 56°C to 62°C were further examined to detect the optimal reaction temperature for HAdV‐MCDA‐One assay. The optimal thermal value for HAdV‐MCDA‐One assay was determined according to the fluorescence value yielded from positive and negative controls. A 7500 real‐time PCR instrument was used to screen an appropriate temperature for subsequent experiments. All experiments were performed in triplicate, and one‐way ANOVA followed by Tukey's test was used for statistical analysis.

2.6. Sensitivity and Specificity Analysis of HAdV‐MCDA‐One

This study utilized the serial dilution method for the evaluation of the sensitivity of HAdV‐MCDA‐One. The standard plasmid (1.59 × 106 copies/μL) was diluted 10‐fold using DW (1.59 × 105 copies/μL, 1.59 × 104 copies/μL, 1.59 × 103 copies/μL, 1.59 × 102 copies/μL, 1.59 × 101 copies/μL, 1.59 copies/μL, and 1.59 × 10−1 copies/μL, respectively). To evaluate the potential impact of clinical sample matrices on assay performance, the same serial dilutions were additionally prepared using HAdV‐negative clinical specimens. Each dilution in both the DW‐based series and the negative clinical matrix series was tested in triplicate to assess assay reproducibility and analytical robustness. Statistical analysis was performed using one‐way ANOVA and Tukey's test to ensure data reliability.

For specificity analysis, 13 non‐HAdV pathogens were chosen for validation, accompanied by the HAdV pathogen and plasmid as positive control, while distilled water functioned as the blank control. Each test was performed with three parallel replicates, and one‐way ANOVA followed by Tukey's test was used for statistical analysis. The specific pathogen species and sources are shown in Table S2.

2.7. Reliability of HAdV‐MCDA‐One Assay in Clinical Testing

Ninety‐six clinical pharyngeal swab samples harvested from patients admitted to the hospital in the Children's Hospital of Capital Institute of Pediatrics were utilized in this study to appraise the reliability of HAdV‐MCDA‐One for clinical sample diagnosis. The protocol for sample collection underwent review and was authorized by Capital Institute of Pediatrics (Ethical approval number: SHERLL2024067). A quantitative real‐time PCR developed by Qiu, F. Z. et al. was chosen as the control method to appraise the performance of HAdV‐ MCDA‐One assay [25]. All nucleic acid extracted from pharyngeal swab samples as templates were involved in two methods and each trial was implemented in three technical replicates.

2.8. Reliability of HAdV‐MCDA‐One Assay in Co‐Infection Conditions

To verify the reliability of the HAdV‐MCDA‐One assay in co‐infection scenarios, three co‐infection samples (Human Rhinovirus, Epstein‐Barr virus, and Influenza A virus each co‐infected with HAdV) were tested in three repetitions under optimal conditions. One‑way ANOVA followed by Tukey's test was used for statistical analysis to ensure the reliability and reproducibility of the results.

3. Results

3.1. Principle of the HAdV‐MCDA‐One Assay

The principle of the HAdV‐MCDA‐One assay is illustrated in Figure 1a. The assay integrates MCDA and CRISPR‐Cas12b for the one‐step detection of HAdV‐3 and HAdV‐7. In this system, MCDA is used for initial nucleic acid amplification, while the CRISPR‐Cas12b system provides secondary signal amplification and readout. To enable CRISPR‐Cas12b detection, the MCDA reaction utilizes a modified primer (CP1*) containing a Cas12b protospacer adjacent motif (PAM) site (5’‐TTC‐3’). Once the CRISPR‐Cas12b/gRNA complex recognizes the genomic target site, directed by the gRNA and the PAM site, Cas12b effector is activated. This activation triggers the trans‐cleavage of single‐stranded DNA (ssDNA) reporter probes, generating a strong fluorescent signal. In the absence of the target, the effector remains inactive, and no fluorescence is generated. Unlike conventional two‐step assays that require transferring amplification products to a separate CRISPR detection mixture (Figure 1b), the HAdV‐MCDA‐One platform performs both reactions in a single tube, eliminating liquid transfer steps and providing a real‐time fluorescence readout (Figure 1c).

Figure 1.

Figure 1

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Schematic diagram of HAdV‐MCDA‐One assay. (a) Schematic illustration of the assay principle. The targets were amplified by MCDA, and the products with PAM site were recognized by the CRISPR/Cas12b system. The trans‐cleavage activity of the Cas12b effector is activated to cleave ssDNA non‐specifically. The fluorescent signal for positive samples (target present) and no signal for negative samples (non‐target) was reported. (b) Steps for traditional CRISPR‐mediated assays, including template preparation (e.g., extraction of DNA from pharyngeal swab samples), MCDA reaction, secondary open tubes to add Cas‐gRNA binary complex to the assay system, and fluorescent signal report. (c) Workflow of the HAdV‐MCDA‐One assay. Template preparation takes ~10 min, followed by the MCDA‐One reaction at 60°C for 50 min, with fluorescence readout to determine positive (rising fluorescence curve) or negative (flat curve) results.

3.2. Optimization of the HAdV‐MCDA‐One Assay Reaction Conditions

To ascertain the optimal reaction temperature, we first tested the MCDA component across a temperature gradient from 58°C to 69°C. The reaction efficiency increased with temperature within this range (Figure 2a). Given that the AapCas12b enzyme is active up to 65°C, we then tested the complete HAdV‐MCDA‐One system at temperature interval of 56°C–62°C. At 60°C, the assay exhibited the highest fluorescence signal in the positive control (Figure 2b) and the lowest background signal in the negative control (Figure S2). Therefore, 60°C was selected as the optimal working temperature and adopted as the standard for all subsequent experimental procedure.

Figure 2.

Figure 2

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Optimum temperature screening. (a) The performance curve of the MCDA reaction (at 58°C–69°C) for the detection of HAdV was obtained by real‐time turbidimetry. Turbidity of > 0.1 was considered to be the positive threshold value. (b) Bar graph of fluorescence values (FAM) by the HAdV‐MCDA‐One assay at different temperatures (56°C–62°C). (c) Fluorescence curves at different temperatures (56°C‐62°C), describing the temperature‐dependent kinetics of the HAdV‐MCDA‐One assay.

3.3. Validation of the HAdV‐MCDA Primer Set

To validate the primer set, amplification was performed at 60°C with HAdV plasmid DNA designated as the positive control, Epstein‐Barr virus DNA as the negative control, and nuclease‐free water as the blank control. A substantial increase in fluorescence was detected only in the reaction containing the HAdV plasmid template, with no significant signal change noted in the negative and blank control samples (Figure 3). These results confirmed the functionality and high specificity of the HAdV‐MCDA primer set.

Figure 3.

Figure 3

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Confirmation of feasibility of the HAdV‐MCDA primer set. (a) Bar graph showing fluorescence values (FAM) for three samples: HAdV Plasmid, Epstein‐Barr Virus, and Nucleic Acid‐free Water. ****p < 0.0001; ns, no significance. (b) Fluorescence kinetic curves over 50 min for HAdV Plasmid, Epstein‐Barr Virus, and Nucleic Acid‐free Water. Only HAdV Plasmid shows a marked fluorescence increase. (c) Heatmap visualization of fluorescence values across time (0–50 min) for the HAdV Plasmid, Epstein‐Barr Virus, and Nucleic Acid‐free Water.

3.4. Sensitivity and Specificity of the HAdV‐MCDA‐One Assay

The analytical sensitivity of the assay was evaluated using 10‐fold serial dilutions of the HAdV plasmid, ranging from 1.59 × 106 to 1.59 × 10−1 copies/μL. Dilutions were prepared in either DW or HAdV‐negative clinical specimens to assess potential matrix effects. The fluorescence signals were consistently detected in reactions containing at least 1.59 copies of plasmid DNA, whereas no signal was observed at lower concentrations or in the corresponding negative controls (Figure 4). Accordingly, the limit of detection was determined to be 1.59 copies per reaction. Comparable detection sensitivity was observed in both DW and clinical matrix backgrounds, although slightly stronger fluorescence signals were obtained from plasmids diluted in DW at the same concentration, suggesting a minor matrix‐associated effect on signal intensity.

Figure 4.

Figure 4

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Sensitivity of HAdV‐MCDA‐One assay. (a) Bar graph of fluorescence values (FAM) for HAdV‐Plasmid at different concentrations (1.59 × 105 copies/μL, 1.59 × 104 copies/μL, 1.59 × 103 copies/μL, 1.59 × 102 copies/μL, 1.59 × 101 copies/μL, 1.59 copies/μL, and 1.59 × 10−1 copies/μL, and DW). ****p < 0.0001; ns, no significance; DW, distilled water. (b) Fluorescence kinetic curves over 50 min for HAdV‐Plasmid at varying concentrations. (c) Heatmap of fluorescence values across time (0–50 min) and concentrations, color intensity corresponds to signal strength. (d) Bar graph of fluorescence values (FAM) for HAdV‐Plasmid at different concentrations (1.59 × 105 copies/μL, 1.59 × 104 copies/μL, 1.59 × 103 copies/μL, 1.59 × 102 copies/μL, 1.59 × 101 copies/μL, 1.59 copies/μL, and 1.59 × 10−1 copies/μL, and negative matrix control). ****p < 0.0001; ns, no significance.(e) Fluorescence kinetic curves over 50 min at varying concentrations diluted by HAdV‐negative clinical specimens. (f) Heatmap of fluorescence values across time (0–50 min) and concentrations diluted by HAdV‐negative clinical specimens, color intensity corresponds to signal strength.

To assess analytical specificity, the assay was tested against a panel of pathogens. A strong fluorescent signal was observed only for the HAdV pathogen and plasmid, with no distinct signal detected for any other pathogens (Figure 5), demonstrating 100% specificity in the detection of HAdV‐3 and HAdV‐7.

Figure 5.

Figure 5

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Specificity of HAdV‐MCDA‐One assay. (a) Bar graph of fluorescence values for multiple viruses. Only HAdV‐Plasmid shows a strong signal, highlighting assay specificity. ****p < 0.0001; ns, no significance. (b) Fluorescence kinetic curves for selected viruses over 50 min. Only HAdV‐Plasmid has a rising curve, confirming specific detection. (c) Heatmap of fluorescence values for various viruses across time.

3.5. Clinical Reliability of the HAdV‐MCDA‐One Assay

With the aim of evaluating the clinical utility of the HAdV‐MCDA‐One assay, 96 pharyngeal swabs were tested, and the results were subjected to comparison with the reference qPCR. A total of 48 positive and 48 negative samples were identified by the HAdV‐MCDA‐One assay (Figure 6a). Notably, we report core diagnostic performance metrics (sensitivity, specificity, PPV, NPV) for qualitative assays (Table 1), which confirm 100% concordance with qPCR (Figure 6b). These metrics quantify the assay's ability to correctly identify HAdV‐3/7 positive and negative samples, supporting its clinical reliability for rapid screening.

Figure 6.

Figure 6

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Reliability of HAdV‐MCDA‐One assay in clinical testing. (a) Heatmap visualizing fluorescence intensity distribution of 96 clinical samples analyzed by the HAdV‐MCDA‐One assay, reflecting detection signals for HAdV in clinical specimens. (b) Heatmap visualizing HAdV‐3 detection results via real‐time fluorescence PCR, providing a comparative analysis with the HAdV‐MCDA‐One assay.

Table 1.

Comparisons of HAdV‐MCDA‐one and real‐time PCR assay.

PCR
HAdV‐ MCDA‐One Positive Negative Sens Spec PPV NPV
Positive 48 0 100% 100% 100% 100%
Negative 0 48
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Note: Diagnostic performance metrics of the HAdV‐MCDA‐One assay (qualitative) compared with real‐time qPCR (reference method). Sensitivity, specificity, PPV, and NPV are reported as standard metrics for evaluating qualitative diagnostic tools.

Abbreviations: NPV, negative prediction value; PPV, positive prediction value; Sens, sensitivity; Spec, specificity.

3.6. Reliability of HAdV‐MCDA‐One Assay in Co‐Infection Conditions

To further confirm the reliability of this assay, we tested three groups of co‐infection samples; no cross‐reactivity or signal interference was observed from the co‐infection pathogens, and all samples showed strong positive fluorescence signals (Figure S3). The HAdV‐MCDA‐One assay exhibited high specificity and stability in co‐infection conditions, making it suitable for clinical samples with potential multiple pathogen infections.

4. Discussion

HAdV, particularly serotypes 3 and 7, remains a significant public health threat. While molecular diagnostics like PCR are the “gold standard,” their operational complexity limits their use in field or resource‐constrained settings. IATs have addressed many of these challenges [26, 27]. Among these, MCDA has shown high sensitivity and specificity in various applications, positioning it as a promising next‐generation IAT [16, 17, 28, 29]. However, most reported MCDA‐CRISPR methods require a two‐step process involving the transfer of amplicons, which increases operational complexity and aerosol‐mediated contamination risk (Figure 1b).

To overcome this limitation, we developed the HAdV‐MCDA‐One assay, a one‐pot platform for the rapid and reliable identification of HAdV‐3 and HAdV‐7. The key innovation is the integration of MCDA amplification and CRISPR‐Cas12b detection within a single reaction, which simplifies the procedure and minimizes contamination risk [30]. The assay provides reliable results after a 50‐min incubation at 60°C, eliminating the need for thermal cycling equipment. By coupling MCDA target amplification with CRISPR‐Cas12b signal amplification, the assay achieves single molecule detection with an LoD of 1.59 copies per reaction, as confirmed by triplicate measurements with consistent positive signals at the lowest concentration. Furthermore, the sequence‐specific recognition by both the MCDA primers and the Cas12b‐gRNA complex provides a dual‐check mechanism that ensures high specificity. Consistent with this design, no cross‐reactivity was observed with the pathogens evaluated in this study. Additionally, sequence alignment analysis also showed that other HAdV serotypes contain multiple mismatches within the primer‐binding and gRNA‐target regions (Figure S1).

The HAdV‐MCDA‐One assay demonstrates several advantages. First, the single‐base mismatch recognition capability of the CRISPR‐Cas12b system ensures accurate target discrimination, effectively eliminating false positives from non‐specific amplification. Second, the use of a fluorescent readout provides an objective result and reduces the variability associated with subjective naked‐eye interpretation of colorimetric assays. Third, as suggested in previous studies, the trans‐cleavage of reporter probes in CRISPR‐based assays provides a secondary signal amplification cascade, contributing to the high sensitivity observed [31].

The current study suffers from several limitations. First, although no cross‐reactivity was observed with the pathogens evaluated in this study, certain genomic regions are conserved among human adenovirus serotypes, limited cross‐detection of non‐target HAdV types cannot be completely excluded. Therefore, the assay is likely to preferentially detect HAdV‐3 and HAdV‐7, while any potential detection of other serotypes may occur with reduced sensitivity. Further evaluation using additional adenovirus serotypes and clinical isolates is needed to comprehensively assess assay specificity and potential serotype‐dependent differences in detection performance. Second, a comprehensive clinical validation was implemented with a limited number of samples from a single source. This may explain why only HAdV‐3 was detected among the positive samples. Future studies should expand to multi‐center clinical cohorts with comprehensive pathogen screening for confirmed mixed infection scenarios, to further validate the robustness of the assay. Third, although the proposed assay demonstrated rapid, sensitive, and highly specific detection of HAdV‐3/7, it was designed primarily as a qualitative tool for rapid screening rather than quantitative viral load measurement, for which real‐time PCR remains more suitable. Nevertheless, rapid qualitative detection may still provide important clinical value for routine screening and early diagnosis, particularly in resource‐limited settings. Fourth, although the assay showed favorable cost‐effectiveness, the estimated costs reported in this study were based on current laboratory‐scale reagent pricing and may vary according to reagent suppliers, procurement scale, regional pricing, and market fluctuations (Table S3). Future optimization and large‐scale manufacturing may further reduce the overall testing cost.

In conclusion, we have established and authenticated the HAdV‐MCDA‐One, an assay that enables ultra‐rapid, ultra‐sensitive, and highly specific detection of HAdV‐3 and −7. This assay is capable of improving diagnostic efficiency, mitigate misdiagnosis, and guide appropriate antimicrobial stewardship. As a powerful complementary diagnostic, the HAdV‐MCDA‐One assay can aid in clinical management and public health surveillance of HAdV infections.

Author Contributions

Linglong Wan performed the experiments, analyzed the data and drafted the manuscript. Juan Zhou supervised and funded this study and revised the manuscript. Lei Yu, Xiaolan Huang, Fei Xiao, Nan Jia, and Min Chen helped in sample preparation and data analysis. Jin Fu and Yu Zhang provided experimental reagents and data collection. Zhaomin Feng conceived this study and revised the manuscript. Yi Wang conceived, funded and supervised this study and revised the manuscript.

Ethics Statement

The studies involving sample collection were reviewed and approved by Capital Institute of Pediatrics (Ethical approval number: SHERLL2024067).

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supporting File

JMV-98-e71032-s001.docx (1.5MB, docx)

Acknowledgments

This research was funded by Beijing Natural Science Foundation (7262007), Beijing Natural Science Foundation‐Changping Innovation Joint Fund Project (Grant Nos. L234047), Research Foundation of Capital Institute of Pediatrics (XZDX‐2025‐002), Beijing research center for respiratory infectious diseases project (BJRID2025‐009; BJRID2026‐011), Pathogen spectrum and host marker analysis in respiratory tract infection of children (Grant 2024‐0040), and Open Project Fund from Key Laboratory of Coal Environmental Pathogenicity and Prevention (Shanxi Medical University), Ministry of Education, China (Grant MEKLCEPP/SXMU‐202412).

Wan L., Zhou J., Yu L., et al., “A One‐Pot CRISPR‐Cas12b Assay for Rapid Detection of Human Adenovirus Serotypes 3 and 7,” Journal of Medical Virology 98 (2026): e71032, 10.1002/jmv.71032.

Linglong Wan, Juan Zhou, and Lei Yu contributed equally to this article.

Contributor Information

Zhaomin Feng, Email: fengzhaomin214@163.com.

Yi Wang, Email: wildwolf0101@163.com.

Data Availability Statement

Data will be made available on request.

References

  • 1. Nemerow G. R., Stewart P. L., and Reddy V. S., “Structure of Human Adenovirus,” Current Opinion in Virology 2 (2012): 115–121. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2. Yu X., Veesler D., Campbell M. G., et al., “Cryo‐EM Structure of Human Adenovirus D26 Reveals the Conservation of Structural Organization Among Human Adenoviruses,” Science Advances 3 (2017): e1602670. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3. Lenaerts L., De Clercq E., and Naesens L., “Clinical Features and Treatment of Adenovirus Infections,” Reviews in Medical Virology 18 (2008): 357–374. [DOI] [PubMed] [Google Scholar]
  • 4. Jin R., Qin T., Li P., et al., “Increased Circulation of Adenovirus in China During 2023‐2024: Association With an Increased Prevalence of Species B and School‐Associated Transmission,” Journal of Infection 90 (2025): 106475. [DOI] [PubMed] [Google Scholar]
  • 5. Lion T., “Adenovirus Infections in Immunocompetent and Immunocompromised Patients,” Clinical Microbiology Reviews 27 (2014): 441–462. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6. Xiao K., Cao Y., Yan P., et al., “A Large‐Scale Single‐Cell Atlas Reveals the Peripheral Immune Panorama of Bacterial Pneumonia,” American Journal of Respiratory and Critical Care Medicine 211, no. 12 (2025): 2363–2381. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7. Xiao K., Cao Y., Han Z., et al., “A Pan‐Immune Panorama of Bacterial Pneumonia Revealed by a Large‐Scale Single‐Cell Transcriptome Atlas,” Signal Transduction and Targeted Therapy 10 (2025): 5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8. Binder A. M., Biggs H. M., Haynes A. K., et al., “Human Adenovirus Surveillance ‐ United States, 2003‐2016,” MMWR. Morbidity and Mortality Weekly Report 66 (2017): 1039–1042. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9. Leland D. S. and Ginocchio C. C., “Role of Cell Culture for Virus Detection in the Age of Technology,” Clinical Microbiology Reviews 20 (2007): 49–78. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10. Fischer C., Jo W. K., Haage V., Moreira‐Soto A., de Oliveira Filho E. F., and Drexler J. F., “Challenges Towards Serologic Diagnostics of Emerging Arboviruses,” Clinical Microbiology and Infection 27, no. 9 (2021): 1221–1229. [DOI] [PubMed] [Google Scholar]
  • 11. Kubista M., Andrade J. M., Bengtsson M., et al., “The Real‐Time Polymerase Chain Reaction,” Molecular Aspects of Medicine 27 (2006): 95–125. [DOI] [PubMed] [Google Scholar]
  • 12. Xiao F., Fu J., Huang X., et al., “Loop‐Mediated Isothermal Amplification Coupled With Nanoparticle‐Based Lateral Flow Biosensor for Monkeypox Virus Detection,” Talanta 269 (2024): 125502. [DOI] [PubMed] [Google Scholar]
  • 13. Zhou J., Xiao F., Fu J., et al., “Rapid, Ultrasensitive and Highly Specific Diagnosis of Mycoplasma Pneumoniae by a Crispr‐Based Detection Platform,” Frontiers in Cellular and Infection Microbiology 13 (2023): 1147142. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14. Chi Z., Wu Y., Chen L., et al., “CRISPR‐Cas14a‐integrated Strand Displacement Amplification for Rapid and Isothermal Detection of Cholangiocarcinoma Associated Circulating MicroRNAs,” Analytica Chimica Acta 1205 (2022): 339763. [DOI] [PubMed] [Google Scholar]
  • 15. Zhou J., Xiao F., Fu J., et al., “Rapid Detection of Monkeypox Virus by Multiple Cross Displacement Amplification Combined With Nanoparticle‐Based Biosensor Platform,” Journal of Medical Virology 95 (2023): e28479. [DOI] [PubMed] [Google Scholar]
  • 16. Xiao F., Zhang Y., Xu W., et al., “Real‐Time Fluorescent Multiple Cross Displacement Amplification for Rapid and Sensitive Mycoplasma Pneumoniae Detection,” Frontiers in Cellular and Infection Microbiology 14 (2024): 1423155. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17. Zhu X., Wang X., Li S., et al., “Rapid, Ultrasensitive, and Highly Specific Diagnosis of COVID‐19 by CRISPR‐Based Detection,” ACS Sensors 6 (2021): 881–888. [DOI] [PubMed] [Google Scholar]
  • 18. Amitai G. and Sorek R., “CRISPR‐Cas Adaptation: Insights into the Mechanism of Action,” Nature Reviews Microbiology 14 (2016): 67–76. [DOI] [PubMed] [Google Scholar]
  • 19. Kaminski M. M., Abudayyeh O. O., Gootenberg J. S., Zhang F., and Collins J. J., “CRISPR‐Based Diagnostics,” Nature Biomedical Engineering 5 (2021): 643–656. [DOI] [PubMed] [Google Scholar]
  • 20. Villiger L., Joung J., Koblan L., Weissman J., Abudayyeh O. O., and Gootenberg J. S., “CRISPR Technologies for Genome, Epigenome and Transcriptome Editing,” Nature Reviews Molecular Cell Biology 25 (2024): 464–487. [DOI] [PubMed] [Google Scholar]
  • 21. Jia N., Wang C., Liu X., et al., “A CRISPR‐Cas12a‐based Platform for Ultrasensitive Rapid Highly Specific Detection of Mycobacterium Tuberculosis in Clinical Application,” Frontiers in Cellular and Infection Microbiology 13 (2023): 1192134. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22. Chen X., Tan Y., Wang S., et al., “A CRISPR‐Cas12b‐Based Platform for Ultrasensitive, Rapid, and Highly Specific Detection of Hepatitis B Virus Genotypes B and C in Clinical Application,” Frontiers in Bioengineering and Biotechnology 9 (2021): 743322. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23. Gootenberg J. S., Abudayyeh O. O., Lee J. W., et al., “Nucleic Acid Detection With CRISPR‐Cas13a/C2c2,” Science 356 (2017): 438–442. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24. Wan L., Huang X., Fu J., et al., “Rapid Detection of Human Adenovirus by Multiple Cross Displacement Amplification Combined With Nanoparticle‐Based Biosensor Platform,” Sensors and Actuators Reports 8 (2024): 100252. [Google Scholar]
  • 25. Qiu F., Shen X., Zhao M., et al., “A Triplex Quantitative Real‐Time PCR Assay for Differential Detection of Human Adenovirus Serotypes 2, 3 and 7,” Virology Journal 15 (2018): 81. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26. Yuan H., Chao Y., and Shum H. C., “Droplet and Microchamber‐Based Digital Loop‐Mediated Isothermal Amplification (dLAMP),” Small 16 (2020): e1904469. [DOI] [PubMed] [Google Scholar]
  • 27. Yin W., Zhuang J., Li J., et al., “Digital Recombinase Polymerase Amplification, Digital Loop‐Mediated Isothermal Amplification, and Digital CRISPR‐Cas Assisted Assay: Current Status, Challenges, and Perspectives,” Small 19 (2023): e2303398. [DOI] [PubMed] [Google Scholar]
  • 28. Sun C., Jia N., Huang X., et al., “Real‐Time Multiple Cross Displacement Amplification Assay for Rapid and Sensitive Detection of Haemophilus Influenzae,” Frontiers in Cellular and Infection Microbiology 12 (2022): 1004183. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29. Huang X., Yu L., Sun C., et al., “Visual and Fluorescent Detection of Moraxella Catarrhalis by Multiple Cross Displacement Amplification‐Based Assay,” Heliyon 10 (2024): e38805. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30. Zhou J., Xiao F., Fu J., et al., “A One‐Pot, One‐Step CRISPR Platform (CRISPR‐One) for Nucleic Acid Detection: Application for the Detection of Mycoplasma Pneumoniae,” Sensors and Actuators B: Chemical 422 (2025): 136600. [Google Scholar]
  • 31. Wan L., Zhou J., Yu L., et al., “Rapid, Highly Specific and Sensitive Detection of Human Adenovirus Using Multiple Cross Displacement Amplification Coupled With CRISPR‐Cas12a‐based Detection,” Talanta Open 11 (2025): 100462. [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supporting File

JMV-98-e71032-s001.docx (1.5MB, docx)

Data Availability Statement

Data will be made available on request.

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