Carrying out pseudo dual nucleic acid detection from sample to visual result in a polypropylene bag with CRISPR/Cas12a

Wu, Hui; Chen, Yanju; Shi, Yaowei; Wang, Liu; Zhang, Mengyao; Wu, Jian; Chen, Huan

doi:10.1016/j.bios.2021.113001

Cited by 36 publications

(24 citation statements)

References 21 publications

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“…Compared to other traditional nucleic acid detection methods, CRISPR technology offers overwhelming advantages in the aspects of detection cost, efficiency, portability, and distinctiveness ( Li et al, 2021 ; Wu et al, 2021 ). However, to improve the detection sensitivity, nucleic acid amplification should be carried out before the CRISPR nucleic acid detection reaction.…”

Section: Introductionmentioning

confidence: 99%

A one-pot CRISPR/Cas13a-based contamination-free biosensor for low-cost and rapid nucleic acid diagnostics

Liu

Zhao

et al. 2022

Biosensors and Bioelectronics

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The pandemic due to the outbreak of 2019 coronavirus disease (COVID-19) caused by novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has raised significant public health concerns. Rapid, affordable, and accurate diagnostic testing not only paves the way for the effective treatment of diseases, but also plays a crucial role in preventing the spreading of infectious diseases. Herein, a one-pot CRISPR/Cas13a-based visual biosensor was proposed and developed for the rapid and low-cost nucleic acid detection. By combining Cas13a cleavage and Recombinase Polymerase Amplification (RPA) in a one-pot reaction in a disposable tube-in-tube vessel, amplicon contamination could be completely avoided. The RPA reaction is carried out in the inner tube containing two hydrophobic holes at the bottom. After the completion of amplification reaction, the reaction solution enters the outer tube containing pre-stored Cas13a reagent under the action of centrifugation or shaking. Inner and outer tubes are combined to form an independent reaction pot to complete the nucleic acid detection without opening the lid. This newly developed nucleic acid detection method not only meets the need of rapid nucleic acid detection at home without the need for any specialized equipment, but also fulfils the requirement of rapid on-site nucleic acid detection with the aid of small automated instruments. In this study, CRISPR/Cas13a and CRISPR/Cas12a were used to verify the reliability of the developed one-pot nucleic acid detection method. The performance of the system was verified by detecting the DNA virus, i.e., African swine fever virus (ASFV) and the RNA virus, i.e., SARS-Cov-2. The results indicate that the proposed method possesses a limit of detection of 3 copy/μL. The negative and positive test results are consistent with the results of real-time fluorescence quantitative polymerase chain reaction (PCR), but the time required is shorter and the cost is lower. Thus, this study makes this method available in resource-limited areas for the purpose of large-scale screening and in case of epidemic outbreak.

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Section: Introductionmentioning

confidence: 99%

A one-pot CRISPR/Cas13a-based contamination-free biosensor for low-cost and rapid nucleic acid diagnostics

Liu

Zhao

et al. 2022

Biosensors and Bioelectronics

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“…The ITP-CRISPR in its current design also requires laboratory-based instruments (such as a source meter and camera-mounted inverted epifluorescence microscope) and the LoD obtained (10 copies/µL) was similar to that achieved with LFD in other CRISPR assays [14,48,56]. Nonetheless, the sample-to-result time of ITP-CRISPR (~35 min), which is inclusive of the RNA extraction step, is still shorter than RNA extraction-free CRISPRbased assays (50-75 min) [60][61][62]. Furthermore, ITP-CRISPR is amenable to automation, miniaturization, and integration of different analytical operations.…”

Section: Other Assay Formatsmentioning

confidence: 67%

“…Wu et al [60] demonstrated how a low-cost polypropylene (PP) bag-based approach may be used to facilitate at-home COVID-19 nucleic acid testing [60]. The three-chamber PP bag was designed to be flexible so that mixing could be performed by pressing the chamber with fingers and a foam is also placed in the lid of the PP bag to enable the device to float on water.…”

Section: Other Assay Formatsmentioning

confidence: 99%

“…The CRISPR-Cas reagent mixture for the target gene is only added at the end of the amplification process and a further incubation at 37 • C for 10 min is required prior to the visualization of fluorescence emission with a portable UV lamp in a dark room. A visible fluorescent signal indicates that the sample is positive, but a negative sample must be verified with a second round of CRISPR-Cas assay for RNase P to rule out a false negative result [60]. The LoD obtained (20 copies/reaction) was the same as that achieved in the tube format with a real-time thermocycler as well as that of the visual-based, one-pot RT-LAMP-CRISPR assay developed by Chen et al [53].…”

Section: Other Assay Formatsmentioning

confidence: 99%

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Harnessing CRISPR-Cas to Combat COVID-19: From Diagnostics to Therapeutics

Chan

Ang

et al. 2021

Life

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The coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), remains a global threat with an ever-increasing death toll even after a year on. Hence, the rapid identification of infected individuals with diagnostic tests continues to be crucial in the on-going effort to combat the spread of COVID-19. Viral nucleic acid detection via real-time reverse transcription polymerase chain reaction (rRT-PCR) or sequencing is regarded as the gold standard for COVID-19 diagnosis, but these technically intricate molecular tests are limited to centralized laboratories due to the highly specialized instrument and skilled personnel requirements. Based on the current development in the field of diagnostics, the programmable clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) system appears to be a promising technology that can be further explored to create rapid, cost-effective, sensitive, and specific diagnostic tools for both laboratory and point-of-care (POC) testing. Other than diagnostics, the potential application of the CRISPR–Cas system as an antiviral agent has also been gaining attention. In this review, we highlight the recent advances in CRISPR–Cas-based nucleic acid detection strategies and the application of CRISPR–Cas as a potential antiviral agent in the context of COVID-19.

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“…In this study, we intended to introduce HCR-based CRISPR-Cas12a into an electrochemical biosensor platform for sensitive and specific detection of S. typhimurium (Scheme 1 ) [ 21 ] Electrochemical biosensors have been widely applied to detect pathogenic bacteria in food safety processes due to their fast signal readouts, simplicity, high sensitivity, and low cost, thus providing a practical or deployable point-of-care system [ 17 ]. The nonspecific ssDNA reporter (methylene blue (MB) tag modified on hairpin DNA (hpDNA)) was drafted with an MB electrochemical tag for signal transduction and a thiol moiety to tether on the sensor surface to acquire the signal electrically.…”

Section: Introductionmentioning

confidence: 99%

Electrochemical biosensor for detecting pathogenic bacteria based on a hybridization chain reaction and CRISPR-Cas12a

Liu

Feng

et al. 2021

Anal Bioanal Chem

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In this study, Lba Cas12a (Cpf1) as one of the CRISPR systems from Lachnospiraceae bacterium was coupled with a hybridization chain reaction (HCR) to develop an electrochemical biosensor for detecting the pathogenic bacterium, Salmonella typhimurium . Autonomous cross-opening of functional DNA hairpin structures of HCR yielded polymer double-stranded DNA wires consisting of numerous single-stranded DNAs, which initiated the trans-cleavage activity of CRISPR-Cas12a to indiscriminately cleave random single-stranded DNA labeling electrochemical tags on the surface of the electrode. It led to a variation in the electron transfer of electrochemical tags. The polymer double-stranded DNA of HCR was immobilized on dynabeads (DBs) via the S. typhimurium aptamer and released from DBs. The established method could selectively and sensitively quantify S. typhimurium in samples with detection limits of 20 CFU/mL. Our study provides a novel insight for exploring universal analytical methods for pathogenic bacteria based on CRISPR-Cas12a coupled with HCR. Supplementary Information The online version contains supplementary material available at 10.1007/s00216-021-03733-6.

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Carrying out pseudo dual nucleic acid detection from sample to visual result in a polypropylene bag with CRISPR/Cas12a

Cited by 36 publications

References 21 publications

A one-pot CRISPR/Cas13a-based contamination-free biosensor for low-cost and rapid nucleic acid diagnostics

A one-pot CRISPR/Cas13a-based contamination-free biosensor for low-cost and rapid nucleic acid diagnostics

Harnessing CRISPR-Cas to Combat COVID-19: From Diagnostics to Therapeutics

Electrochemical biosensor for detecting pathogenic bacteria based on a hybridization chain reaction and CRISPR-Cas12a

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