Recent advances in recombinase polymerase amplification: Principle, advantages, disadvantages and applications

Frontiers in Cellular and Infection Microbiology, Год журнала: 2022, Номер 12

Опубликована: Ноя. 28, 2022

After the outbreak of SARS-CoV-2, nucleic acid testing quickly entered people’s lives. In addition to polymerase chain reaction (PCR) which was commonly used in testing, isothermal amplification methods were also important methods. Among several common like displaced amplification, rolling circle and so on, recombinase (RPA) recently paid more attention to. It had advantages a simple operation, fast speed, at 37-42°C, et al. So it very suitable for field detection. However, there still some disadvantages RPA. Herein, our review mainly summarized principle, advantages, The specific applications RPA bacterial detection, fungi virus parasite drug resistance gene genetically modified food SARS-CoV-2 detection described. hoped that latest research progress on could be better delivered readers who interested

Язык: Английский

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Light‐Start CRISPR‐Cas12a Reaction with Caged crRNA Enables Rapid and Sensitive Nucleic Acid Detection

Angewandte Chemie International Edition, Год журнала: 2023, Номер 62(23)

Опубликована: Апрель 5, 2023

Abstract The clustered regularly interspaced short palindromic repeats (CRISPR) system is a promising platform for nucleic acid detection. Regulating the CRISPR reaction would be extremely useful to improve detection efficiency and speed of diagnostic applications. Here, we have developed light‐start CRISPR‐Cas12a by employing caged RNA (crRNA). When combined with recombinase polymerase amplification, robust photocontrolled one‐pot assay achieved. simpler 50‐fold more sensitive than conventional assay. This improved also facilitates development faster method. clinical samples demonstrated that 10–20 min sufficient effective detection, which much current gold‐standard technique PCR. We expect this advance in diagnostics promote its widespread applications biomedicine, agriculture, food safety.

Язык: Английский

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Ultrasensitive single-step CRISPR detection of monkeypox virus in minutes with a vest-pocket diagnostic device

Nature Communications, Год журнала: 2024, Номер 15(1)

Опубликована: Апрель 16, 2024

Abstract The emerging monkeypox virus (MPXV) has raised global health concern, thereby highlighting the need for rapid, sensitive, and easy-to-use diagnostics. Here, we develop a single-step CRISPR-based diagnostic platform, termed SCOPE (Streamlined CRISPR On Pod Evaluation platform), field-deployable ultrasensitive detection of MPXV in resource-limited settings. viral nucleic acids are rapidly released from rash fluid swab, oral saliva, urine samples 2 min via streamlined lysis protocol, followed by 10-min recombinase polymerase amplification (RPA)-CRISPR/Cas13a reaction. A pod-shaped vest-pocket analysis device achieves whole process reaction execution, signal acquisition, result interpretation. can detect as low 0.5 copies/µL (2.5 copies/reaction) within 15 sample input to answer. We validate developed assay on 102 clinical male patients / volunteers, testing results 100% concordant with real-time PCR. single-molecular level sensitivity minutes simplified procedure performed miniaturized wireless device, which is expected spur substantial progress enable practice application diagnostics techniques point-of-care setting.

Язык: Английский

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CRISPR/Cas12a-derived ratiometric fluorescence sensor for high-sensitive Pb2+ detection based on CDs@ZIF-8 and DNAzyme

Biosensors and Bioelectronics, Год журнала: 2024, Номер 251, С. 116089 - 116089

Опубликована: Фев. 3, 2024

Язык: Английский

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Universal crRNA Acylation Strategy for Robust Photo‐Initiated One‐Pot CRISPR–Cas12a Nucleic Acid Diagnostics

Angewandte Chemie International Edition, Год журнала: 2024, Номер 63(23)

Опубликована: Апрель 2, 2024

Abstract Spatiotemporal regulation of clustered regularly interspaced short palindromic repeats (CRISPR) system is attractive for precise gene editing and accurate molecular diagnosis. Although many efforts have been made, versatile efficient strategies to control CRISPR are still desirable. Here, we proposed a universal accessible acylation strategy regulate the CRISPR–Cas12a by 2′‐hydroxyls (2′‐OH) on crRNA strand with photolabile agents (PLGs). The introduction PLGs confers suppression function rapid restoration reaction upon light exposure regardless sequences. Based this strategy, constructed PhotO‐Initiated Robust One‐pot Testing (POIROT) platform integrated recombinase polymerase amplification (RPA), which showed two orders magnitude more sensitive than conventional one‐step assay comparable two‐step assay. For clinical sample testing, POIROT achieved high‐efficiency detection performance gold‐standard quantitative PCR (qPCR) in sensitivity specificity, but faster qPCR method. Overall, believe will promote development other photo‐controlled technologies one‐pot assay, even expand applications fields controllable CRISPR‐based genomic editing, disease therapy, cell imaging.

Язык: Английский

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CRISPR-Cas-based techniques for pathogen detection: Retrospect, recent advances, and future perspectives

Journal of Advanced Research, Год журнала: 2022, Номер 50, С. 69 - 82

Опубликована: Окт. 30, 2022

Early detection of pathogen-associated diseases are critical for effective treatment. Rapid, specific, sensitive, and cost-effective diagnostic technologies continue to be challenging develop. The current gold standard pathogen detection, polymerase chain reaction technology, has limitations such as long operational cycles, high cost, technician instrumentation requirements.This review examines highlights the technical advancements CRISPR-Cas in provides an outlook future development, multi-application scenarios, clinical translation.Approaches enabling nucleic acids that highly cheap, portable necessary. CRISPR-Cas9 specificity targeting "collateral cleavage" activity CRISPR-Cas12/Cas13/Cas14 show significant promise acid technology. These methods have a specificity, versatility, rapid cycle. In this paper, CRISPR-Cas-based discussed depth. Although CRISPR-Cas-mediated solutions face challenges, their powerful capabilities will pave way ideal tools.

Язык: Английский

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Integrating CRISPR-Cas12a into a Microfluidic Dual-Droplet Device Enables Simultaneous Detection of HPV16 and HPV18

Analytical Chemistry, Год журнала: 2023, Номер 95(6), С. 3476 - 3485

Опубликована: Фев. 1, 2023

Fast, simplified, and multiplexed detection of human papillomaviruses (HPVs) is great importance for both clinical management population screening. However, current HPV methods often require sophisticated instruments laborious procedures to detect multiple targets. In this work, we developed a simple microfluidic dual-droplet device (M-D3) the simultaneous HPV16 HPV18 by combining CRISPR-Cas12a system recombinase polymerase amplification (RPA) assay. A new approach pressure/vacuum was proposed efficient droplet generation with minimal sample consumption. Two groups droplets that separately encapsulate relevant Cas12a/crRNA fluorescent green or red reporters are parallelly generated, followed automatic imaging discriminate subtypes based on specific fluorescence droplets. The M-D3 platform performs high sensitivity (∼0.02 nM unamplified plasmids) specificity in detecting DNA. By RPA Cas12a assay, allows on-chip DNA simultaneously within 30 min, reaching limit 10–18 M (∼1 copy/reaction). Moreover, outstanding performance validated testing 20 patient samples infection risk, showing 92.3% 100%. integrating generator, CRISPR-Cas12a, RPA, provides an way two most harmful holds potential applications nucleic acid testing.

Язык: Английский

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Fluorescence and Colorimetric Analysis of African Swine Fever Virus Based on the RPA-Assisted CRISPR/Cas12a Strategy

Analytical Chemistry, Год журнала: 2023, Номер 95(20), С. 8063 - 8069

Опубликована: Май 11, 2023

It is well-established that different detection modes are necessary for corresponding applications, which can effectively reduce matrix interference and improve the accuracy. Here, we reported a magnetic separation method based on recombinase polymerase amplification (RPA)-assisted clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a dual-mode analysis of African swine fever virus (ASFV) genes, including colorimetry fluorescence. The ASFV gene was selected as initial RPA template to generate amplicon. amplicon then recognized by CRISPR-associated RNA (crRNA), activating trans-cleavage activity Cas12a leading nonspecific cleavage ssDNA well significant release alkaline phosphatase (ALP) in ALP-ssDNA modified bead. released ALP catalyze para-nitrophenyl phosphate para-nitrophenol, resulting substantial changes absorbance fluorescence, both be used with naked eye. This strategy allows sensitive DNA, 20 copies/mL limit; no cross-reactivity other viruses observed. A good linear relationship obtained serum. In addition, this sensor displayed 100% specificity sensitivity clinical sample analysis. integrates high fluorescence easy readout enables simple, low-cost, highly viral nucleic acid, thereby providing broad prospect practical application diagnosis infection.

Язык: Английский

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One-Pot Assay for Rapid Detection of Benzimidazole Resistance in Venturia carpophila by Combining RPA and CRISPR/Cas12a

Journal of Agricultural and Food Chemistry, Год журнала: 2023, Номер 71(3), С. 1381 - 1390

Опубликована: Янв. 10, 2023

High resistance to benzimidazole fungicides in Venturia carpophila is caused by the point mutation E198K of β-tubulin (TUB2) gene. Traditional methods for detection fungicide are time-consuming, which routinely based on tedious operation, reliance expensive equipment, and specially trained people. Therefore, it important establish efficient field V. make suitable management strategies ensure food safety. Based recombinase polymerase amplification (RPA) combined with CRISPR/Cas12a, a rapid one-pot assay ORCas12a-BRVc (one-pot RPA-CRISPR/Cas12 platform) was established carpophila. The enabled adding components at bottom wall tube separately, solving problems aerosol contamination decreased sensitivity competing DNA substrates between Cas12a cleavage RPA amplification. could accomplish minimum 7.82 × 103 fg μL-1V. genomic 45 min 37 °C. Meanwhile, this showed excellent specificity due specific recognition ability Cas12a-crRNA complex. Further, we method that rapidly extract from within 2 achieve more simple fields. has advantages simplicity, rapidity, high sensitivity, specificity, ease operation without need precision instruments isolate culture pathogens. This first application platform combination CRISPR/Cas12a can be used monitoring resistant populations fields, providing guidance making peach scab.

Язык: Английский

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CRISPR gel: A one-pot biosensing platform for rapid and sensitive detection of HIV viral RNA

Analytica Chimica Acta, Год журнала: 2023, Номер 1262, С. 341258 - 341258

Опубликована: Апрель 24, 2023

Язык: Английский

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