One-tube RPA-CRISPR Cas12a/Cas13a rapid detection of methicillin-resistant Staphylococcus aureus

Liu, Yujie; Liu, Hui; Yu, Guanliu; Sun, Wenbo; Aizaz, Muhammad; Yang, Guiwen; Chen, Lei

doi:10.1016/j.aca.2023.341757

Cited by 24 publications

(8 citation statements)

References 31 publications

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“…For instance, when employing Cas12a/Cas13a, the presence of cis -cleavage activity can adversely affect amplification efficiency, making it challenging to achieve a one-pot approach, and the cumulative errors introduced through multiple steps are non-negligible. 133,134…”

Section: Discussionmentioning

confidence: 99%

Cas-based bacterial detection: recent advances and perspectives

Lan,

Shu,

Jiang

et al. 2024

Analyst

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Section: Discussionmentioning

confidence: 99%

Cas-based bacterial detection: recent advances and perspectives

Lan,

Shu,

Jiang

et al. 2024

Analyst

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“…( An et al, 2021 ), Neisseria gonorrhoeae ( Allan-Blitz et al, 2023 ) and Group B Streptococci (GBS; Li Z. et al, 2023 ). Moreover, the CRISPR-Cas13a technology was also utilized to identify multiple genes, such as toxin genes in Clostridioides difficile ( Jiang et al, 2023 ), the mecA and clfA genes in methicillin-resistant S. aureus (MRSA; Liu et al, 2023 ), the blaKPC gene in Enterobacterales ( Liang et al, 2023 ), the lcrV gene in Yersinia pestis ( Schultzhaus et al, 2021 ). Similarly, in this study, we established a one-tube and two-step RPA-Cas13a platform ( Figure 1 ) to detect the mexX gene from P. aeruginosa , with respectable sensitivity and reasonable specificity.…”

Section: Discussionmentioning

confidence: 99%

“…To reduce viscosity, some groups employed one lyophilized pellet from the DNA constant temperature rapid amplification kit, which yielded approximately five individual reactions ( Miao et al, 2023 ). In addition, a novel one-tube method SHERCOK was also exploited to deal with the viscosity ( Xiong et al, 2022 ; Liu et al, 2023 ). Although this method reduces the risk of sample contamination and maintains detection sensitivity ( Xiong et al, 2022 ; Liu et al, 2023 ), it is essentially a two-step method of SHERCOK in a strict sense.…”

Section: Discussionmentioning

confidence: 99%

“…In addition, a novel one-tube method SHERCOK was also exploited to deal with the viscosity ( Xiong et al, 2022 ; Liu et al, 2023 ). Although this method reduces the risk of sample contamination and maintains detection sensitivity ( Xiong et al, 2022 ; Liu et al, 2023 ), it is essentially a two-step method of SHERCOK in a strict sense. In subsequent work, we will investigate strategies to overcome the problems given by enzyme incompatibility and the crowding effect of one-tube molecular detection, thereby making the one-step RPA-Cas13a determination method a simple, dependable, and stable diagnostic tool.…”

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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“…These technologies generally require a duration of 2 to 4 days for the outcomes. Therefore, various culture-independent techniques have been devised to expedite the identification of MRSA, such as enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) [ 5 - 7 ]. Nevertheless, the application of these methods targeting mecA gene detection is still restricted by high cost, low accuracy, time-consuming, complicated sample pre-treatment (complicated gene extraction procedures).…”

Section: Introductionmentioning

confidence: 99%

Allosteric Probe-Based Colorimetric Assay for Direct Identification and Sensitive Analysis of Methicillin Resistance of Staphylococcus aureus

Chu,

Zhao

2024

J. Microbiol. Biotechnol.

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The accurate and rapid detection of methicillin-resistance of Staphylococcus aureus (SA) holds significant clinical importance. However, the methicillin-resistance detection strategies commonly require complicated cell lysis and gene extraction. Herein, we devised a novel colorimetric approach for the sensitive and accurate identification of methicillin-resistance of SA by combining allosteric probe-based target recognition with self-primer elongation-based target recycling. The PBP2a aptamer in the allosteric probe successfully identified the target MRSA, leading to the initiation of self-primer elongation based-cascade signal amplification. The peroxidase-like hemin/G-quadruplex undergo an isothermal autonomous process that effectively catalyzes the oxidation of ABTS 2- and produces a distinct blue color, enabling the visual identification of MRSA at low concentrations. The method offers a shorter duration for bacteria cultivation compared to traditional susceptibility testing methods, as well as simplified manual procedures for gene analysis. The overall amplification time for this test is 60 min, and it has a detection limit of 3 CFU/ml. In addition, the approach has exceptional selectivity and reproducibility, demonstrating commendable performance when tested with real samples. Due to its advantages, this colorimetric assay exhibits considerable potential for integration into a sensor kit, thereby offering a viable and convenient alternative for the prompt and on-site detection of MRSA in patients with skin and soft tissue infections.

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One-tube RPA-CRISPR Cas12a/Cas13a rapid detection of methicillin-resistant Staphylococcus aureus

Cited by 24 publications

References 31 publications

Cas-based bacterial detection: recent advances and perspectives

Cas-based bacterial detection: recent advances and perspectives

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

Allosteric Probe-Based Colorimetric Assay for Direct Identification and Sensitive Analysis of Methicillin Resistance of Staphylococcus aureus

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