A CRISPR/Cas12a Based Universal Lateral Flow Biosensor for the Sensitive and Specific Detection of African Swine-Fever Viruses in Whole Blood

Wu, Jinghua; Mukama, Omar; Wu, Wei; Li, Zhiyuan; Habimana, Jean de Dieu; Zhang, Yinghui; Zeng, Rong; Nie, Chengrong; Zeng, Lingwen

doi:10.3390/bios10120203

Cited by 33 publications

(22 citation statements)

References 25 publications

(36 reference statements)

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“…Compared with traditional PCR-based methods, the proposed CRISPR-based assay exhibited cleavage activity on the target DNA, indicating that it can reduce cross-contamination in the laboratory. Moreover, the amplification condition of 37 °C also reduced the loss and contamination caused by high temperatures during PCR [ 37 ]. In addition, the PCR-based methods required more than 1.5 h to complete, while the CE–RAA–CRISPR method provided results in less than 1 h. Application of the assay to real food samples showed accurate and consistent detection results compared with qPCR.…”

Section: Discussionmentioning

confidence: 99%

CE–RAA–CRISPR Assay: A Rapid and Sensitive Method for Detecting Vibrio parahaemolyticus in Seafood

Cao²,

Zhang

et al. 2022

Foods

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Vibrio parahaemolyticus is one of the major pathogenic Vibrio species that contaminate seafood. Rapid and accurate detection is crucial for avoiding foodborne diseases caused by pathogens and is important for food safety management and mariculture. In this study, we established a system that combines chemically enhanced clustered regularly interspaced short palindromic repeats (CRISPR) and recombinase-aided amplification (RAA) (CE–RAA–CRISPR) for detecting V. parahaemolyticus in seafood. The method combines RAA with CRISPR-associated protein 12a (Cas12a) for rapid detection in a one-pot reaction, effectively reducing the risk of aerosol contamination during DNA amplifier transfer. We optimized the primers for V. parahaemolyticus, determined the optimal crRNA/Cas12a ratio, and demonstrated that chemical additives (bovine serum albumin and L-proline) could enhance the detection capacity of Cas12a. The limit of detection (at optimal conditions) was as low as 6.7 × 101 CFU/mL in pure cultures and 7.3 × 101 CFU/g in shrimp. Moreover, this method exhibited no cross-reactivity with other microbial pathogens. The CE–RAA–CRISPR assay was compared with the quantitative polymerase chain reaction assay using actual food samples, and it showed 100% diagnostic agreement.

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Section: Discussionmentioning

confidence: 99%

CE–RAA–CRISPR Assay: A Rapid and Sensitive Method for Detecting Vibrio parahaemolyticus in Seafood

Cao²,

Zhang

et al. 2022

Foods

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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

143

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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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“…In socioeconomic terms, CRISPR-based biosensing techniques were also developed to satisfy the demand from animal husbandry, agriculture, and forestry. For example, a Cas12a-based lateral flow biosensor combined with PCR amplification was used for the detection of the African swine fever virus (ASFV), achieving a sensitivity of 2.5 × 10 –15 M within 2 h from swine blood ( Wu et al, 2020 ). Another Cas12a-based reversible valve-assisted chip was established for the rapid detection of V. parahaemolyticus for the seafood test, with an LoD of 30 copies/reaction by using 600 μl of samples ( Wu et al, 2021b ).…”

Section: Applications Of Crispr-based Biosensing Techniquesmentioning

confidence: 99%

Powerful CRISPR-Based Biosensing Techniques and Their Integration With Microfluidic Platforms

Chen

et al. 2022

Front. Bioeng. Biotechnol.

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In the fight against the worldwide pandemic coronavirus disease 2019 (COVID-19), simple, rapid, and sensitive tools for nucleic acid detection are in urgent need. PCR has been a classic method for nucleic acid detection with high sensitivity and specificity. However, this method still has essential limitations due to the dependence on thermal cycling, which requires costly equipment, professional technicians, and long turnover times. Currently, clustered regularly interspaced short palindromic repeats (CRISPR)-based biosensors have been developed as powerful tools for nucleic acid detection. Moreover, the CRISPR method can be performed at physiological temperature, meaning that it is easy to assemble into point-of-care devices. Microfluidic chips hold promises to integrate sample processing and analysis on a chip, reducing the consumption of sample and reagent and increasing the detection throughput. This review provides an overview of recent advances in the development of CRISPR-based biosensing techniques and their perfect combination with microfluidic platforms. New opportunities and challenges for the improvement of specificity and efficiency signal amplification are outlined. Furthermore, their various applications in healthcare, animal husbandry, agriculture, and forestry are discussed.

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A CRISPR/Cas12a Based Universal Lateral Flow Biosensor for the Sensitive and Specific Detection of African Swine-Fever Viruses in Whole Blood

Cited by 33 publications

References 25 publications

CE–RAA–CRISPR Assay: A Rapid and Sensitive Method for Detecting Vibrio parahaemolyticus in Seafood

CE–RAA–CRISPR Assay: A Rapid and Sensitive Method for Detecting Vibrio parahaemolyticus in Seafood

CRISPR-Cas based virus detection: Recent advances and perspectives

Powerful CRISPR-Based Biosensing Techniques and Their Integration With Microfluidic Platforms

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