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1.
Biosens Bioelectron ; 216: 114683, 2022 Nov 15.
Article in English | MEDLINE | ID: mdl-36088673

ABSTRACT

Developing highly accurate and simple approaches to rapidly identify and isolate SARS-CoV-2 infected patients is important for the control of the COVID-19 pandemic. We, herein, reported the performance of a Cas12a-assisted RTF-EXPAR strategy for the identification of SARS-CoV-2 RNA. This assay combined the advantages of RTF-EXPAR with CRISPR-Cas12a can detect SARS-CoV-2 within 40 min, requiring only isothermal control. Particularly, the simultaneous use of EXPAR amplification and CRISPR improved the detection sensitivity, thereby realizing ultrasensitive SARS-CoV-2 RNA detection with a detection limit of 3.77 aM (∼2 copies/µL) in an end-point fluorescence read-out fashion, and at 4.81 aM (∼3 copies/µL) level via a smartphone-assisted analysis system (RGB analysis). Moreover, Cas12a increases the specificity by intrinsic sequence-specific template recognition. Overall, this method is fast, sensitive, and accurate, needing minimal equipment, which holds great promise to meet the requirements of point-of-care molecular detection of SARS-CoV-2.


Subject(s)
Biosensing Techniques , COVID-19 , Biosensing Techniques/methods , COVID-19/diagnosis , CRISPR-Cas Systems/genetics , Humans , Nucleic Acid Amplification Techniques/methods , Pandemics , RNA, Viral/analysis , RNA, Viral/genetics , SARS-CoV-2/genetics , Sensitivity and Specificity
2.
Biosens Bioelectron ; 211: 114393, 2022 Sep 01.
Article in English | MEDLINE | ID: mdl-35609450

ABSTRACT

A fast and simple Cas13a-based assay approach for direct detecting Ebola RNA in unamplified samples is reported. The procedure (named Cas-Roller) is comprised of a 10-min Cas13a-mediated cleavage protocol, followed by a DNA roller running for 30 min. This involves Cas13a collateral cleaving a suitably designed substrate in the presence of Ebola virus RNA sequence, and the cleavage product is used for DNA roller to amplify and generate fluorescent signals. After optimization of the conditions, the assay is able to achieve a limit of detection as low as 291 aM (∼175 copies RNA/µL) along with excellent anti-interfering performance in human serum and blood detection, which is ∼310-fold improved compared with the direct CRISPR assay. The entire workflow can be completed in ∼40 min at 37 °C without any pre-amplification, transcription, or centrifugation steps, thus avoiding the generation of false-negative or positive results. In addition, the downstream roller reaction is independent of the target sequence, this method can be applied to detect any other RNA by merely redesigning the hybridization regions of the crRNA. Overall, this strategy gives a new idea for the construction of simple and accurate Cas13a-based assays for the direct detection of RNA.


Subject(s)
Biosensing Techniques , Hemorrhagic Fever, Ebola , CRISPR-Cas Systems/genetics , DNA , Hemorrhagic Fever, Ebola/diagnosis , Hemorrhagic Fever, Ebola/genetics , Humans , RNA
3.
Biosens Bioelectron ; 176: 112954, 2021 Mar 15.
Article in English | MEDLINE | ID: mdl-33412428

ABSTRACT

CRISPR/Cas system have drawn increasing attention in accurate and sensitive nucleic acids detection. Herein, we reported a novel Cas12a-based electrochemiluminescence biosensor for target amplification-free human papilloma virus subtype (HPV-16) DNA detection. During this detection process, Cas12a employed its two-part recognition mechanism to improve the specificity and trans-cleavage capability to achieve signal amplification, while L-Methionine stabilized gold nanoclusters (Met-AuNCs) were served as high-efficiency ECL emitters to achieve ECL signal transition. Given the unique combination of Cas12a with ECL technique, the detection limit was determined as 0.48 pM and the whole detection could be completed within 70 min. We also validated the practical application of the proposed biosensor by using undiluted human blood samples, which gives impetus to the design of new generations of CRISPR/Cas detection system beyond the traditional ones with ultimate applications in sensing analysis and diagnostic technologies.


Subject(s)
Biosensing Techniques , CRISPR-Cas Systems , DNA/genetics , Gold , Humans , Nucleic Acid Amplification Techniques
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