Split T7 promoter-based isothermal transcription amplification for one-step fluorescence detection of SARS-CoV-2 and emerging variants

Yoon, Taehwi; Shin, Jiye; Choi, Hyun‐Jung; Park, Ki Soo

doi:10.1016/j.bios.2022.114221

Cited by 38 publications

(21 citation statements)

References 30 publications

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“…The recent advent of SARS-CoV-2 and its variants have threatened humanity, incurring a high social cost and emphasized the importance of nucleic acid testing. Great research efforts have been devoted to detecting nucleic acids with high sensitivity and selectivity that could contribute to the effective control and prevention of viral transmission [1][2][3].…”

Section: Introductionmentioning

confidence: 99%

Highly Efficient DNA Reporter for CRISPR/Cas12a-Based Specific and Sensitive Biosensor

et al. 2022

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In addition to cis-cleavage activity that recognizes and cleaves nucleic acid sequences, a trans-cleavage activity that indiscriminately and non-specifically cleaves single-stranded DNA or RNA has been discovered in some Cas proteins, including Cas12a and Cas13a. Various detection methods using this activity have been widely reported. Herein, we describe a new highly efficient DNA reporter (5′-TTATT-CCCCC-3′; TTATT-5C) that outperformed the existing AT-rich DNA reporter (5′-TTATT-3′) used in most Cas12a-based target nucleic detection assays. By systematically investigating the effect of DNA reporter length and sequence on the trans-cleavage activity of Cas12a, we achieved up to a 100-fold increase in fluorescence signal intensity derived from the trans-cleavage activity of Cas12a compared to that achieved using the existing AT-rich DNA reporter. The new DNA reporter was also applied, along with the existing AT-rich DNA reporter, for the detection of the Salmonella enterotoxin ( stn ) gene. Importantly, both detection speed and limit were significantly enhanced with the new DNA reporter. In addition, polymerase chain reaction (PCR) was adopted to the CRISR/Cas-Based system of the new DNA reporter, thereby confirming its practical applicability. The high-efficiency DNA reporter described herein can pave the way for further improving the trans-cleavage activity of other Cas proteins, as well as the sensitivity of CRISPR/Cas-Based systems. Supplementary Information The online version contains supplementary material available at 10.1007/s13206-022-00081-0.

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Section: Introductionmentioning

confidence: 99%

Highly Efficient DNA Reporter for CRISPR/Cas12a-Based Specific and Sensitive Biosensor

et al. 2022

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“…It is possible to limit the use of enzymes for the light-up aptamer-encoding 3WJ technology to only RNA polymerase. When this paper was in preparation, a similar system was reported, in which DNA polymerase to produce the promotor sequence was avoided due to the use of a split T7 promoter ( Yoon et al, 2022 ). In this case, the antisense promoter sequence was included in the same probe that also encoded a light-up aptamer (analogue of the TP component herein).…”

Section: Discussionmentioning

confidence: 99%

Single-tube isothermal label-free fluorescent sensor for pathogen detection based on genetic signatures

Reed

Gerasimova

2022

Front. Chem.

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We report on a single-tube biosensor for real-time detection of bacterial pathogens with multiplex capabilities. The biosensor consists of two DNA probes, which bind to the complementary fragment of a bacterial RNA to form a three-way junction (3WJ) nucleic acid structure. One of the probes encodes a fluorescent light-up RNA aptamer under T7 promoter. It allows for generation of multiple aptamer copies due to elongation and transcription of the 3WJ structure in the presence of the complementary target. The aptamer coordinates and thereby enhances fluorescence of a cognate fluorogenic dye, allowing for fluorescent detection of the RNA target. Multiple aptamer copies can be produced from a single target-dependent 3WJ structure allowing for amplification and visual observation of the signal. The limit of detection depended on the assay time and was found to be 1.7 nM or 0.6 nM for 30-min or 60-min assay, respectively, when N-methylmesoporphyrin IX (NMM) was used as a fluorescent indicator. The sensor is excellent in analyzing folded RNA targets and differentiating between closely related sequences due to the multicomponent character of the target-interrogating probe. Response to unamplified samples of total bacterial RNA from Mycobacterium tuberculosis complex or Escherichia coli was observed with excellent selectivity within 30 min under isothermal conditions at 50°C in a one-tube one-step assay. Several bacterial species can be detected in multiplex by utilizing biosensors with the template strands encoding different light-up aptamers. The isothermal one-tube-one-step format of the assay and the possibility to monitor the signal visually makes it amenable to use in a point-of-care scenario.

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“…Primer sequences used for the detection of SARS-CoV-2, SARS-CoV, HCoV-OC43; Table S2: Price comparison between the commercial product and the introduced method for colorimetric detection of viral RNA; Figure S1 Refs. [34][35][36] are cited in Supplementary Materials. Institutional Review Board Statement: Not applicable.…”

Section: Supplementary Materialsmentioning

confidence: 99%

A Rotatable Paper Device Integrating Reverse Transcription Loop-Mediated Isothermal Amplification and a Food Dye for Colorimetric Detection of Infectious Pathogens

2022

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In this study, we developed a rotatable paper device integrating loop-mediated isothermal amplification (RT-LAMP) and a novel naked-eye readout of the RT-LAMP results using a food additive, carmoisine, for infectious pathogen detection. Hydroxyl radicals created from the reaction between CuSO4 and H2O2 were used to decolor carmoisine, which is originally red. The decolorization of carmoisine can be interrupted in the presence of DNA amplicons produced by the RT-LAMP reaction due to how DNA competitively reacts with the hydroxyl radicals to maintain the red color of the solution. In the absence of the target DNA, carmoisine is decolored, owing to its reaction with hydroxyl radicals; thus, positive and negative samples can be easily differentiated based on the color change of the solution. A rotatable paper device was fabricated to integrate the RT-LAMP reaction with carmoisine-based colorimetric detection. The rotatable paper device was successfully used to detect SARS-CoV-2 and SARS-CoV within 70 min using the naked eye. Enterococcus faecium spiked in milk was detected using the rotatable paper device. The detection limits for the SARS-CoV-2 and SARS-CoV targets were both 103 copies/µL. The rotatable paper device provides a portable and low-cost tool for detecting infectious pathogens in a resource-limited environment.

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Split T7 promoter-based isothermal transcription amplification for one-step fluorescence detection of SARS-CoV-2 and emerging variants

Cited by 38 publications

References 30 publications

Highly Efficient DNA Reporter for CRISPR/Cas12a-Based Specific and Sensitive Biosensor

Highly Efficient DNA Reporter for CRISPR/Cas12a-Based Specific and Sensitive Biosensor

Single-tube isothermal label-free fluorescent sensor for pathogen detection based on genetic signatures

A Rotatable Paper Device Integrating Reverse Transcription Loop-Mediated Isothermal Amplification and a Food Dye for Colorimetric Detection of Infectious Pathogens

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