Accurate MRSA identification through dual-functional aptamer and CRISPR-Cas12a assisted rolling circle amplification

Xu, Lin; Dai, Qingqing; Shi, Zhanying; Liu, Xiaotao; L, Gao; Wang, Zhengzheng; Zhu, Xiaoyun; Li, Zhen

doi:10.1016/j.mimet.2020.105917

Cited by 68 publications

(43 citation statements)

References 20 publications

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“…Later that year, Liang et al [ 139 ] successfully extended a similar integrated approach to liposomal aptamer-targeting of CRISPR/Cas9 against vascular endothelial growth factor A in mouse models of osteosarcoma. In 2020, Xu et al [ 140 ] reported the first example of the integration of dual aptamer technology and CRISPR-Cas12a [ 137 ]-assisted rolling circle amplification to obtain both accurate identification and highly-sensitive detection of methicillin-resistant Staphylococcus ureus (MRSA), detection of which is a global priority.…”

Section: Discussionmentioning

confidence: 99%

Recent advances in aptamer applications for analytical biochemistry

Zon

2022

Analytical Biochemistry

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Aptamers are typically defined as relatively short (20–60 nucleotides) single-stranded DNA or RNA molecules that bind with high affinity and specificity to various types of targets. Aptamers are frequently referred to as “synthetic antibodies” but are easier to obtain, less expensive to produce, and in several ways more versatile than antibodies. The beginnings of aptamers date back to 1990, and since then there has been a continual increase in aptamer publications. The intent of the present account was to focus on recent original research publications, i.e., those appearing in 2019 through April 2020, when this account was written. A Google Scholar search of this recent literature was performed for relevance-ranking of articles. New methods for selection of aptamers were not included. Nine categories of applications were organized and representative examples of each are given. Finally, an outlook is offered focusing on “faster, better, cheaper” application performance factors as key drivers for future innovations in aptamer applications.

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Section: Discussionmentioning

confidence: 99%

Recent advances in aptamer applications for analytical biochemistry

Zon

2022

Analytical Biochemistry

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“…Given that antibody-based systems are generated using IgG antibodies which are the main antibodies used in the immune response, PA is not ideal for antibody-based detection systems; however, it could be used as a target for an aptamer-based system. Penicillin binding protein 2a (PBP2a) is also a conserved protein found only on MRSA strains, allowing for further specificity of detection [91]. Some bacteria, such as C. difficile, conserve non-host evasion surface proteins.…”

Section: Detection Systemsmentioning

confidence: 99%

“…Another example where aptamers have been used to detect bacteria, specifically S. aureus, utilized two aptamers, one binding to PA, a unique and conserved protein found on all strains of S. aureus, and one aptamer binding to PBP2a, a protein found only on MRSA [24,33,53,91]. The PA aptamer was conjugated with streptavidin magnetic beads to capture any bacteria expressing PA [56], which includes both MRSA and standard S. aureus.…”

Section: Detection Systemsmentioning

confidence: 99%

“…Once the blocker is released it binds to the padlock completing the circular DNA complex allowing for activation of RCA using Cas12a. This activation then allows a CRISPR enzyme to cleave the product which contains Cy3, unquenching the fluorescent molecule [91]. Aptamers can also be used in label free detection as certain nucleic acid dyes can be used to determine if the aptamers have bound to a target.…”

Section: Detection Systemsmentioning

confidence: 99%

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Novel Detection of Nasty Bugs, Prevention Is Better than Cure

Strom

Crowley

Shigdar

2020

IJMS

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Hospital-acquired infections (HAIs) are a growing concern around the world. They contribute to increasing mortality and morbidity rates and are an economic threat. All hospital patients have the potential to contract an HAI, but those with weakened or inferior immune systems are at highest risk. Most hospital patients will contract at least one HAI, but many will contract multiple ones. Bacteria are the most common cause of HAIs and contribute to 80–90% of all HAIs, with Staphylococcus aureus, Clostridium difficile, Escherichia coli, Acinetobacter baumannii, Pseudomonas aeruginosa and Klebsiella pneumoniae accounting for the majority. Each of these bacteria are highly resistant to antibiotics and can produce a protective film, known as a biofilm, to further prevent their eradication. It has been shown that by detecting and eradicating bacteria in the environment, infection rates can be reduced. The current methods for detecting bacteria are time consuming, non-specific, and prone to false negatives or false positives. Aptamer-based biosensors have demonstrated specific, time-efficient and simple detection, highlighting the likelihood that they could be used in a similar way to detect HAI-causing bacteria.

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“…20 On the basis of this discovery, they reported and applied Cas12a protein by combining recombinase polymerase amplification (RPA) to develop a rapid and accurate test for the detection and classification of human papillomavirus (HPV) in clinical specimens. 20 Based on the ssDNase property of the Cas12a protein, it expands upon the diagnostic applications range of infectious and noninfectious diseases in various situations, such as the detection of virus (i. e. pandemic COVID-19 21 , SARS-CoV-2 22 , and African swine fever virus 23 ) and bacteria 24 (i. e. Staphylococcus aureus, 25 Listeria monocytogenes, 26 Mycobacterium tuberculosis 27 , and Methicillin-resistant Staphylococcus aureus (MRSA) 28 ), it can also be used for pathogen detection in agricultural 29 and aquatic community. 30 CRISPR technology is expected to become a rapid, accurate and portable diagnostic tool in the future.…”

Section: Introductionmentioning

confidence: 99%

Rapid and Visible RPA-Cas12a fluorescence Assay for Accurate Detection of Zoonotic Dermatophytes

Wang

Cai

et al. 2021

Preprint

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Background: Dermatophytosis is an infectious disease of global significance caused by several fungal species, which affects the hair, nails, or superficial layers of the skin. The most common zoonotic dermatophytes are Microsporum canis, Nannizzia gypsea and Trichophyton mentagrophytes. Wood's lamp examination, microscopic identification and fungal culture are the main conventional diagnostic methods used in clinics. Less common methods are dermatophyte PCR and biopsy/histopathology. However, these methods also have limitations for providing both accuracy and timely on-site detection. The recent development of CRISPR-based diagnostic platform provides the possibility of a rapid, accurate, and portable diagnostic tool, which has huge potential for clinical applications. Objectives: The purpose of this study is to establish a molecular method for rapid and accurate diagnosis of clinical dermatophytes, which can accelerate clinical diagnostic testing and help timely treatment. Methods: In this paper, we design a Cas12a-based assay combined with recombinase polymerase amplification (RPA) to differentiate three main zoonotic dermatophytes. The limit of detection (LOD) is determined by using standard strains. A total of 25 clinical samples (hair and scurf) are identified to evaluate the sensitivity and specificity of this assay. Results: The RPA-Cas12a method showed high sensitivity and specificity (100% and 100%, respectively). The results could be observed directly by naked-eyes, and all tested samples were consistent with fungal culture and sequencing results. Conclusions: Compared with other methods, the RPA-Cas12a-fluorescence assay requires less time (30 minutes) and less complicated equipment, and visible changes can be clearly observed, which is suitable for on-site clinical diagnosis.

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Accurate MRSA identification through dual-functional aptamer and CRISPR-Cas12a assisted rolling circle amplification

Cited by 68 publications

References 20 publications

Recent advances in aptamer applications for analytical biochemistry

Recent advances in aptamer applications for analytical biochemistry

Novel Detection of Nasty Bugs, Prevention Is Better than Cure

Rapid and Visible RPA-Cas12a fluorescence Assay for Accurate Detection of Zoonotic Dermatophytes

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