An amplification-free ultra-sensitive electrochemical CRISPR/Cas biosensor for drug-resistant bacteria detection

Suea‐Ngam, Akkapol; Howes, Philip D.; deMello, Andrew J.

doi:10.1039/d1sc02197d

Cited by 88 publications

(50 citation statements)

References 42 publications

(56 reference statements)

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“…Although the RAA-Cas12a method we established has high sensitivity and specificity, it still has several weaknesses. First, our method does not detect the few MRSA strains containing the mecA-like gene (mecC) (Lakhundi and Zhang, 2018), which has also been observed in other molecular strategies (Buchan et al, 2015;Xu et al, 2020;Suea-Ngam et al, 2021). mecC is a new resistance gene recently discovered and identified in MRSA isolated from animals.…”

Section: Discussionmentioning

confidence: 89%

Rapid and Ultrasensitive Detection of Methicillin-Resistant Staphylococcus aureus Based on CRISPR-Cas12a Combined With Recombinase-Aided Amplification

Wang

Liang

et al. 2022

Front. Microbiol.

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Staphylococcus aureus is one of the main pathogens causing hospital and community-acquired infections, in particular, infections caused by methicillin-resistant Staphylococcus aureus (MRSA) cause a higher mortality rate than those caused by methicillin-sensitive strains, which poses a serious global public health problem. Therefore, rapid and ultrasensitive detection of patients with clinical MRSA infection and timely control of infection are essential. Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) based on nucleic acid detection methods are well-known for its high specificity and sensitivity and programmability. Here, we successfully proposed a method based on CRISPR-Cas12a combined with recombinase-aided amplification (RAA) through fluorescent readout to achieve accurate identification and highly sensitive detection of MRSA in clinical samples. Results showed that the limit of detection (LoD) of the RAA-Cas12a method could reach 10 copies/μl at 60 min of reaction. Specificity tests showed that the method could distinguish MRSA from clinically common bacteria. The results of RAA-Cas12a were consistent with that of antimicrobial susceptibility tests (AST) and polymerase chain reaction (PCR) in 83 clinical samples. These results indicated that the detection method based on RAA-Cas12a has high sensitivity and specificity, and provides important value for rapid detection of MRSA.

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Section: Discussionmentioning

confidence: 89%

Rapid and Ultrasensitive Detection of Methicillin-Resistant Staphylococcus aureus Based on CRISPR-Cas12a Combined With Recombinase-Aided Amplification

Wang

Liang

et al. 2022

Front. Microbiol.

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“…Meanwhile, most of the studies reported the LoD in CFU/mL, of which seven studies showed LoD of 1 CFU/mL. Five studies reported the LoD in molarity [ 12 , 17 , 18 , 28 , 38 , 39 ]. Of these, CAS-EXPAR for the detection of Listeria monocytogenes showed the lowest LoD up to the attomolar level [ 38 ].…”

Section: Resultsmentioning

confidence: 99%

CRISPR-Cas Systems-Based Bacterial Detection: A Scoping Review

et al. 2022

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Recently, CRISPR-Cas system-based assays for bacterial detection have been developed. The aim of this scoping review is to map existing evidence on the utilization of CRISPR-Cas systems in the development of bacterial detection assays. A literature search was conducted using three databases (PubMed, Scopus, and Cochrane Library) and manual searches through the references of identified full texts based on a PROSPERO-registered protocol (CRD42021289140). Studies on bacterial detection using CRISPR-Cas systems that were published before October 2021 were retrieved. The Critical Appraisal Skills Programme (CASP) qualitative checklist was used to assess the risk of bias for all the included studies. Of the 420 studies identified throughout the search, 46 studies that met the inclusion criteria were included in the final analysis. Bacteria from 17 genera were identified utilising CRISPR-Cas systems. Most of the bacteria came from genera such as Staphylococcus, Escherichia, Salmonella, Listeria, Mycobacterium and Streptococcus. Cas12a (64%) is the most often used Cas enzyme in bacterial detection, followed by Cas13a (13%), and Cas9 (11%). To improve the signal of detection, 83% of the research exploited Cas enzymes’ trans-cleavage capabilities to cut tagged reporter probes non-specifically. Most studies used the extraction procedure, whereas only 17% did not. In terms of amplification methods, isothermal reactions were employed in 66% of the studies, followed by PCR (23%). Fluorescence detection (67%) was discovered to be the most commonly used method, while lateral flow biosensors (13%), electrochemical biosensors (11%), and others (9%) were found to be less commonly used. Most of the studies (39) used specific bacterial nucleic acid sequences as a target, while seven used non-nucleic acid targets, including aptamers and antibodies particular to the bacteria under investigation. The turnaround time of the 46 studies was 30 min to 4 h. The limit of detection (LoD) was evaluated in three types of concentration, which include copies per mL, CFU per mL and molarity. Most of the studies used spiked samples (78%) rather than clinical samples (22%) to determine LoD. This review identified the gap in clinical accuracy evaluation of the CRISPR-Cas system in bacterial detection. More research is needed to assess the diagnostic sensitivity and specificity of amplification-free CRISPR-Cas systems in bacterial detection for nucleic acid-based tests.

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“…Finally, we note that our model and assay design principles may be broadly applicable to other detection methods such as those which rely on electrochemical impedance measurements for target detection. [23][24][25] Fig. 4.…”

Section: Discussionmentioning

confidence: 99%

Fundamental limits of amplification-free CRISPR-Cas12 and Cas13 diagnostics

Huyke

Ramachandran

Bashkirov³

et al. 2022

Preprint

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Interest in CRISPR diagnostics continues to increase. CRISPR-Cas12 and Cas13 based detection are particularly interesting as they enable highly specific detection of nucleic acids. The fundamental sensitivity limits of Cas12 and Cas13 enzymes are governed by their kinetic rates and are critical to develop amplification-free assays. However, these kinetic rates remain poorly understood and their reporting has been inconsistent. We here measure kinetic parameters for several enzymes (LbCas12a, AsCas12a, AapCas12b, LwaCas13a and LbuCas13a) and evaluate their limits of detection (LoD) for amplification-free target detection. Collectively, we here present quantitation of enzyme kinetics for 14 gRNAs and nucleic acid targets for a total of 50 sets of kinetic rate parameters and 25 LoDs. Importantly, we also validate the self-consistency our measurements by comparing trends and limiting behaviors with a Michaelis-Menten, trans-cleavage reaction kinetics model. Our measurements reveal that activated Cas12 and Cas13 enzymes exhibit typical trans-cleavage catalytic efficiencies between order 105 and 106 M-1 s-1. Moreover, for assays that use fluorescent reporter molecules (ssDNA and ssRNA) for target detection, we find most CRISPR enzymes have an amplification-free LoD in the picomolar range. We find also that successful detection of target requires cleavage (by activated CRISPR enzyme) of at least ~0.1% of the fluorescent reporter molecules. This fraction of cleaved reporters is required to differentiate signal from background, and we hypothesize that this fraction is largely independent of the detection method (i.e., endpoint vs reaction velocity). Our results provide a map of the feasible application range and highlight areas of improvement for the emerging field of CRISPR diagnostics.

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An amplification-free ultra-sensitive electrochemical CRISPR/Cas biosensor for drug-resistant bacteria detection

Abstract: An amplification-free electrochemical CRISPR/Cas biosensor utilizing silver metallization (termed E-Si-CRISPR) allows detection of methicillin-resistant Staphylococcus aureus (MRSA) with excellent sensitivity and specificity.

Cited by 88 publications

References 42 publications

Rapid and Ultrasensitive Detection of Methicillin-Resistant Staphylococcus aureus Based on CRISPR-Cas12a Combined With Recombinase-Aided Amplification

Rapid and Ultrasensitive Detection of Methicillin-Resistant Staphylococcus aureus Based on CRISPR-Cas12a Combined With Recombinase-Aided Amplification

CRISPR-Cas Systems-Based Bacterial Detection: A Scoping Review

Fundamental limits of amplification-free CRISPR-Cas12 and Cas13 diagnostics

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