Cas12a-assisted RTF-EXPAR for accurate, rapid and simple detection of SARS-CoV-2 RNA

Hang, Xiao-Min; Wang, Hui-Yi; Liu, Peng-Fei; Zhao, Kai-Ren; Wang, Li

doi:10.1016/j.bios.2022.114683

Cited by 19 publications

(11 citation statements)

References 50 publications

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“…CRISPR-based pathogen diagnostic tools have ushered in an era of rapid POC detection with high sensitivity and specificity [7], [16]. Integrating CRISPR with isothermal DNA amplification techniques like RPA and Loop-mediated amplification (LAMP) has resulted in detection sensitivity upto attomolar levels [17], [18]. Our CRISPR-Cas12a based detection is able to specifically detect monkeypox virus with an LOD of 60 copies.…”

Section: Resultsmentioning

confidence: 99%

CRISPR/Cas12a assisted specific detection of monkeypox virus

Singh

Misra

Bindal

et al. 2022

Preprint

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ObjectiveTo identify monkeypox-specific sequence and distinguish it from related orthopoxviruses followed by development of a CRISPR-Cas12a-based, specific and sensitive detection of monkeypox virus.MethodsA common detection mixture (CDM) was constituted comprising of CRISPR RNAs (crRNAs) for general orthopoxviruses and monkeypox virus-specific targets along with LbCas12a and fluorescent reporter. Recombinase polymerase amplification (RPA) of target loci was carried out followed by detection using the CRISPR-Cas12 CDM complex. Fluorescence-based read out can be monitored by a fluorescent reader and alternatively can also be visualized under a blue light illuminator by naked eye.ResultsMonkeypox-specific conserved sequences were identified inpolAgene which differ by a single nucleotide polymorphism (SNP) from all the viruses present in genus Orthopoxvirus. Our Cas12a-based assay was capable of specifically distinguishing monkeypox virus from other related orthopoxviruses with an LOD of 60 copies in 1 hour after the initiation of the reaction.ConclusionOur assay exhibits sensitive and specific detection of monkeypox virus which can prove to be of practical value for surveillance in areas infected with multiple orthopoxviruses, especially in hotspots of monkeypox virus infections.

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Section: Resultsmentioning

confidence: 99%

CRISPR/Cas12a assisted specific detection of monkeypox virus

Singh

Misra

Bindal

et al. 2022

Preprint

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“…11,22 Integrating CRISPR with isothermal DNA amplification techniques like RPA and Loop-mediated amplification has resulted in detection sensitivity up to attomolar levels. 23,24 Previously, a few reports have demonstrated CRISPR-based detection of mpox virus. This study differs from the previous reports in two aspects; single copy detection for two genes/targets was not reported 20,21,25,26 and few reports have considered only a single locus for detection.…”

Section: Conclusion and Discussionmentioning

confidence: 99%

“…CRISPR‐based pathogen diagnostic tools have ushered in an era of rapid POC detection with high sensitivity and specificity 11,22 . Integrating CRISPR with isothermal DNA amplification techniques like RPA and Loop‐mediated amplification has resulted in detection sensitivity up to attomolar levels 23,24 . Previously, a few reports have demonstrated CRISPR‐based detection of mpox virus.…”

Section: Conclusion and Discussionmentioning

confidence: 99%

CRISPR‐Cas12a assisted specific detection of mpox virus

Singh

Misra

Bindal

et al. 2023

Journal of Medical Virology

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Mpox virus, a member of genus Orthopoxvirus, causes rash and flu‐like symptoms in humans. In the recent global outbreak, it was reported from several geographical areas that have not historically reported mpox. Point of care, sensitive and specific mpox diagnostic assays are critical in checking the spread of the disease. We have developed a clustered regularly interspaced short palindromic repeats associated Cas12a nuclease‐based assay for detecting mpox virus. Mpox specific conserved sequences were identified in polA (E9L) gene which differ by a single nucleotide polymorphism (SNP) from all the viruses present in the genus Orthopoxvirus. This SNP was exploited in our assay to specifically distinguish mpox virus from other related orthopox viruses with a limit of detection of 1 copy/μl in 30 min. The assay exhibits a sensitive and specific detection of mpox virus which can prove to be of practical value for its surveillance in areas infected with multiple orthopox viruses, especially in hotspots of mpox virus infections.

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“…[25][26][27][28][29] Considering the simple primer design, exponential amplification kinetics, and high amplification efficiency, the EXPAR has attracted considerable attention. 30,31 In the EXPAR, short ssDNA (single-stranded DNA) can trigger a combination of polymerase strand extension and single-strand nicking, so the "extension-cleavagestrand displacement" can be repeated continuously and result in amplification. 32,33 Moreover, the product of the DNA walker reaction, ssDNA, can be a trigger of the EXPAR, which achieves dual signal amplification.…”

Section: Introductionmentioning

confidence: 99%