Development and clinical implications of a novel CRISPR-based diagnostic test for pulmonary Aspergillus fumigatus infection

Li, Zhengtu; Wang, Mingdie; Teng, Xu; Zhan, Yangqing; Chen, Fengyi; Yao, Lin; Li, Shaoqiang; Cheng, Jing; Yan, Feng

doi:10.1016/j.jmii.2021.11.008

Cited by 12 publications

(9 citation statements)

References 22 publications

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“…Up to now, this platform has been used to detect a number of viruses, such as syndrome coronavirus 2 (SARS-CoV-2; Cao et al, 2022 ; López-Valls et al, 2022 ; Su et al, 2022 ) and coronavirus disease (COVID-19). Meanwhile, CRISPR-Cas13a was also used to identify a number of bacterial strains, including Aspergillus fumigatus ( Li et al, 2022 ), Staphylococcus aureus ( Zhou et al, 2020 ), Salmonella spp. ( An et al, 2021 ), Neisseria gonorrhoeae ( Allan-Blitz et al, 2023 ) and Group B Streptococci (GBS; Li Z. et al, 2023 ).…”

Section: Discussionmentioning

confidence: 99%

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Rapid detection of mexX in Pseudomonas aeruginosa based on CRISPR-Cas13a coupled with recombinase polymerase amplification

Zhu,

Wang,

et al. 2024

Front. Microbiol.

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The principal pathogen responsible for chronic urinary tract infections, immunocompromised hosts, and cystic fibrosis patients is Pseudomonas aeruginosa, which is difficult to eradicate. Due to the extensive use of antibiotics, multidrug-resistant P. aeruginosa has evolved, complicating clinical therapy. Therefore, a rapid and efficient approach for detecting P. aeruginosa strains and their resistance genes is necessary for early clinical diagnosis and appropriate treatment. This study combines recombinase polymerase amplification (RPA) and clustered regularly interspaced short palindromic repeats-association protein 13a (CRISPR-Cas13a) to establish a one-tube and two-step reaction systems for detecting the mexX gene in P. aeruginosa. The test times for one-tube and two-step RPA-Cas13a methods were 5 and 40 min (including a 30 min RPA amplification reaction), respectively. Both methods outperform Quantitative Real-time Polymerase Chain Reactions (qRT-PCR) and traditional PCR. The limit of detection (LoD) of P. aeruginosa genome in one-tube and two-step RPA-Cas13a is 10 aM and 1 aM, respectively. Meanwhile, the designed primers have a high specificity for P. aeruginosa mexX gene. These two methods were also verified with actual samples isolated from industrial settings and demonstrated great accuracy. Furthermore, the results of the two-step RPA-Cas13a assay could also be visualized using a commercial lateral flow dipstick with a LoD of 10 fM, which is a useful adjunt to the gold-standard qRT-PCR assay in field detection. Taken together, the procedure developed in this study using RPA and CRISPR-Cas13a provides a simple and fast way for detecting resistance genes.

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Section: Discussionmentioning

confidence: 99%

“…To detect smoke mold, this two-step RPA-SHERLOCK method is also used. Compared to other molds, only the genes of tobacco mold and the plasmid DNA containing target sequences can be detected with higher specificity ( Li et al, 2022 ). There was no discernible difference between the PCR and CRISPR methods ( Li et al, 2022 ).…”

Section: Discussionmentioning

confidence: 99%

Rapid detection of mexX in Pseudomonas aeruginosa based on CRISPR-Cas13a coupled with recombinase polymerase amplification

Zhu,

Wang,

et al. 2024

Front. Microbiol.

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“…Newer techniques such as clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) system may be a promising future technique for diagnosing fungal infections. Recently, Li, Zhengtu et al developed a CRISPR-Cas-based diagnostic test for diagnosing pulmonary Aspergillus fumigatus infection using the ITS region for designing CRISPR Cas RNA (crRNA) and recombinase polymerase amplification (RPA) primer sequence with the T7 promoter [ 98 ].…”

Section: Future Aspects and Challengesmentioning

confidence: 99%

Nucleic-Acid-Based Molecular Fungal Diagnostics: A Way to a Better Future

Gudisa,

Harchand,

Rudramurthy

2024

Diagnostics

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The world has seen a tremendous increase in the number of fungal infections during the past two decades. Recently, the World Health Organisation released the pathogen priority list for fungal infections, signifying the importance of these infections in the fields of research and public health. Microbiology laboratories demand an upgrade in the diagnostic system to keep up with the increased burden of these infections. Diagnosis of fungal infections using conventional techniques has always faced limitations in terms of specificity, sensitivity, and turnaround time. Although these methods are the core pillars of the diagnosis, there is an increased need for molecular approaches. Molecular techniques have revolutionised the field of fungal diagnostics. The diverse array of molecular techniques, including techniques like Polymerase Chain Reaction (PCR), have emerged as a cornerstone in fungal diagnostics. Molecular techniques have transformed fungal diagnostics, providing powerful tools for the rapid and accurate identification of pathogens. As these technologies continue to evolve, their integration into routine clinical practice holds the promise of improving patient outcomes through timely and targeted antifungal interventions. This review will cover the molecular approaches involved in fungal diagnostics, moving from the basic techniques to the advanced-level nucleic-acid-based molecular approaches providing a high throughput and decreased turnaround time for the diagnosis of serious fungal infections.

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“…Additionally, a one-pot CRISPR/Cas13a assay was developed to detect dsDNA of Chlamydia trachomatis with an LOD of 10 fM ( Huang et al, 2021 ). Recently, RPA-SHERLOCK was used to detect Aspergillus fumigatus , with an LOD of 3 copies/L within a total time of 70 min ( Li et al, 2021 ). In addition, Zhan et al combined transcription-mediated amplification (TMA) with Cas13a detection system and used this method to detect mitochondrial large subunit ribosomal RNA of Pneumocystis jirovecii, with an LOD of 2 copies/μl ( Zhan et al, 2022 ).…”

Section: Cas13a-based Detection Of Bacteria and Other Pathogensmentioning

confidence: 99%

CRISPR-Cas13a system: A novel tool for molecular diagnostics

Zhao

Qiu

et al. 2022

Front. Microbiol.

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The clustered regularly interspaced short palindromic repeats (CRISPR) system is a natural adaptive immune system of prokaryotes. The CRISPR-Cas system is currently divided into two classes and six types: types I, III, and IV in class 1 systems and types II, V, and VI in class 2 systems. Among the CRISPR-Cas type VI systems, the CRISPR/Cas13a system has been the most widely characterized for its application in molecular diagnostics, gene therapy, gene editing, and RNA imaging. Moreover, because of the trans-cleavage activity of Cas13a and the high specificity of its CRISPR RNA, the CRISPR/Cas13a system has enormous potential in the field of molecular diagnostics. Herein, we summarize the applications of the CRISPR/Cas13a system in the detection of pathogens, including viruses, bacteria, parasites, chlamydia, and fungus; biomarkers, such as microRNAs, lncRNAs, and circRNAs; and some non-nucleic acid targets, including proteins, ions, and methyl groups. Meanwhile, we highlight the working principles of some novel Cas13a-based detection methods, including the Specific High-Sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK) and its improved versions, Cas13a-based nucleic acid amplification-free biosensors, and Cas13a-based biosensors for non-nucleic acid target detection. Finally, we focus on some issues that need to be solved and the development prospects of the CRISPR/Cas13a system.

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Development and clinical implications of a novel CRISPR-based diagnostic test for pulmonary Aspergillus fumigatus infection

Cited by 12 publications

References 22 publications

Rapid detection of mexX in Pseudomonas aeruginosa based on CRISPR-Cas13a coupled with recombinase polymerase amplification

Rapid detection of mexX in Pseudomonas aeruginosa based on CRISPR-Cas13a coupled with recombinase polymerase amplification

Nucleic-Acid-Based Molecular Fungal Diagnostics: A Way to a Better Future

CRISPR-Cas13a system: A novel tool for molecular diagnostics

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