Application of CRISPR-Cas12a Enhanced Fluorescence Assay Coupled with Nucleic Acid Amplification for the Sensitive Detection of African Swine Fever Virus

Tao, Dagang; Li, Jiajia; Nie, Xiongwei; Xu, Bingrong; Tran-Thi, Thuy-Nhien; Niu, Lili; Liu, Xiangdong; Ruan, Jinxue; Lan, Xiaochen; Peng, Guiqing; Sun, Limeng; Ma, Yunlong; Li, Xinyun; Li, Congcong; Zhao, Shuhong; Xie, Shengsong

doi:10.1021/acssynbio.0c00057

Cited by 65 publications

(57 citation statements)

References 42 publications

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“…Abudayyeh et al, 2021 , Cong et al, 2013 , Gao and Giuffrida, 2018 , Garneau et al, 2010 , Kebschull and Zador, 2015 , Patchsung et al, 2020a , Tao et al, 2020 , Wu et al, 2020 , Xiong et al, 2020 .…”

Section: Uncited Referencementioning

confidence: 99%

CRISPR-Cas based virus detection: Recent advances and perspectives

Yin¹,

Man²,

et al. 2021

Biosensors and Bioelectronics

158

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Viral infections are one of the most intimidating threats to human beings. One convincing example is the coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2. Rapid, sensitive, specific and field-deployable identification of causal viruses is critical for disease surveillance, control and treatment. The shortcomings of current methods create an impending need for developing novel biosensing platforms. CRISPR-Cas systems, especially CRISPR-Cas12a and CRISPR-Cas13a, characterized by their sensitivity, specificity, high base resolution and programmability upon nucleic acid recognition, have been repurposed for molecular diagnostics, surging a new path forward in biosensing. They, as the core of some robust diagnostic tools, are revolutionizing the way that virus can be detected. This review focuses on recent advances in virus detection with CRISPR-Cas systems especially CRISPR-Cas12a/Cas13a. We started with a short introduction to CRISPR-Cas systems and the properties of Cas12a and Cas13a effectors, and continued with reviewing the current advances of virus detection utilizing CRISPR-Cas systems. The significance and advantages of such methods were then discussed. Finally, the challenges and perspectives were proposed. We tried to provide readers with a concise profile of emerging and fast-expanding CRISPR-Cas based biosensing technology, and highlighted its potential applications in a range of scenarios with regard to virus detection.

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Section: Uncited Referencementioning

confidence: 99%

CRISPR-Cas based virus detection: Recent advances and perspectives

Yin¹,

Man²,

et al. 2021

Biosensors and Bioelectronics

158

View full text Add to dashboard Cite

show abstract

“…Considering our result showing effective detection of SARS-CoV-2, we next developed a fast, sensitive, and reliable RAVI-CRISPR-based assay for ASFV using a highly active crRNA identified in our previous study 37 . The strategy for ASFV detection was similar to that for SARS-CoV-2 except that a standard LAMP for DNA amplification was used rather than RT-LAMP since ASFV is a DNA virus (Fig.…”

Section: Asfv Detection By the Ravi-crispr Assaymentioning

confidence: 99%

“…Five crRNAs targeting SARS-CoV-2 E and nine crRNAs targeting SARS-CoV-2 N genes were designed by CRISPR-offinder software 48 (Supplementary Table S10) and complemented to the target sites with a 5ʹ "TTTN" PAM in the DNA strand opposite the target sequence (Supplementary Table S10). A highly active crRNA against ASFV p72 gene was selected from our previous study 37 .…”

Section: Design Of Target Sites For Virus-specific Crrnasmentioning

confidence: 99%

“…Moreover, some studies have reported that Cas-based detection methods can distinguish differences at the single nucleotide level by integrating an optimized CRISPR RNA (crRNA) 29,30,33 , and several improvements to CRISPR-based detection have been developed for POCT applications, such as CRISPR-based lateral flow strip assay 34,35 , CRISPR-based fluorescent cleavage assay 36,37 , plasmonic CRISPR/Cas12a assay 38,39 , and Cas12a-modulated fluorescence resonance energy transfer assay that incorporates nanomaterials for nucleic acid sensing 40 . In addition, mobile phone applications (apps) and mobile phone-based devices have been developed to support the evaluation of lateral flow strips or fluorescence signal read-outs 41,42,43 .…”

Section: Introductionmentioning

confidence: 99%

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Rapid visual CRISPR assay: a naked-eye colorimetric detection method for nucleic acids based on CRISPR/Cas12a and convolutional neural network

Xie

Tao

et al. 2021

Preprint

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Rapid diagnosis based on naked-eye colorimetric detection remains challenging, but it could build new capacities for molecular point-of-care testing (POCT). In this study, we evaluated the performance of 16 types of single-stranded DNA-fluorophore-quencher (ssDNA-FQ) reporters for use with CRISPR/Cas12a based visual colorimetric assays. Among them, 9 ssDNA-FQ reporters were found to be suitable for direct visual colorimetric detection, with especially very strong performance using ROX-labeled reporters. We optimized the reaction concentrations of these ssDNA-FQ reporters for naked-eye read-out of assay results (no transducing component required for visualization). Subsequently, we developed a convolutional neural network algorithm standardize and to automate the analytical colorimetric assessment of images and integrated this into the MagicEye mobile phone software. A field-deployable assay platform named RApid VIsual CRISPR (RAVI-CRISPR) based on a ROX-labeled reporter with isothermal amplification and CRISPR/Cas12a targeting was established. We deployed RAVI-CRISPR in a single tube towards an instrument-less colorimetric POCT format that requires only a portable rechargeable hand warmer for incubation. The RAVI-CRISPR was successfully used for the single-copy detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and African swine fever virus (ASFV). Our study demonstrates this novel RAVI-CRISPR system for distinguishing different pathogenic nucleic acid targets with high specificity and sensitivity as the simplest-to-date platform for rapid pen-side testing.

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“…However, despite PCR and qPCR being the gold standard for ASFV detection in the laboratory, they require thermal cycling instruments and skilled operators, which is not ideal for resource-limited situations. In addition, other molecular diagnostic methods, including the invader assay (14), loop-mediated isothermal ampli cation (15,16), recombinase polymerase ampli cation (17), and methods of detecting ASFV antigens based on the CRISPR system (18, 19), have been developed. Although these methods exhibit high sensitivity and speci city, the majority of them are time-consuming, laborious and costly, which are limitations that seriously hinder their clinical application.…”

Section: Introductionmentioning

confidence: 99%

Horseradish peroxidase-conjugated-nanobody-based blocking ELISA for the rapid and sensitive clinical detection of African swine fever virus antibodies

Zhao

Ren

et al. 2021

Preprint

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Background African swine fever (ASF), which is caused by the ASF virus (ASFV), is a highly contagious hemorrhagic disease that affects pigs and has the potential to cause mortality in almost 100% of domestic pigs and wild boars. Due to the lack of an effective vaccine, the control of ASF must depend on early, efficient, cost-effective detection and strict control and elimination strategies. Traditional molecular and serological testing methods are generally associated with high testing costs, complex operations and high technical requirements. As a promising alternative diagnostic tool to traditional antibodies, nanobodies (Nb) have the advantages of simpler and faster generation, good stability and solubility, and high affinity and specificity. The application of Nbs in the detection of ASFV antibodies in the serum has not yet been reported, to the best of our knowledge. Results Using a phage display technology, one specific Nb against the ASFV p54 protein that exhibited high specificity and affinity to the protein, Nb83, was successfully screened. Nb83 was labeled with horseradish peroxidase (HRP) to create an Nb83-HRP fusion protein in 293T cells. Following the optimization of the reaction conditions, the Nb83-HRP fusion protein was successfully used to establish a blocking enzyme-linked immunosorbent assay (ELISA) to detect ASFV-specific antibodies in pig serum, for the first time. The cutoff value for the blocking ELISA was 39.16%. A total of 210 serum samples were tested using the developed blocking ELISA and a commercial ELISA kit. The specificity of the blocking ELISA was 100%, and the limit of detection was 1:5,120 in inactivated ASFV antibody-positive reference serum samples, with the coincidence rate between the two methods being 98.57%. Conclusions A specific, sensitive and repeatable blocking ELISA was successfully developed based on the unique Nb as a probe, providing a promising method for the detection of anti-ASFV antibodies in clinical pig serum.

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Application of CRISPR-Cas12a Enhanced Fluorescence Assay Coupled with Nucleic Acid Amplification for the Sensitive Detection of African Swine Fever Virus

Cited by 65 publications

References 42 publications

CRISPR-Cas based virus detection: Recent advances and perspectives

CRISPR-Cas based virus detection: Recent advances and perspectives

Rapid visual CRISPR assay: a naked-eye colorimetric detection method for nucleic acids based on CRISPR/Cas12a and convolutional neural network

Horseradish peroxidase-conjugated-nanobody-based blocking ELISA for the rapid and sensitive clinical detection of African swine fever virus antibodies

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