Abstract:13The mortality rate of hemorrhagic African swine fever (ASF), which targets domestic pigs and is caused 14 by African swine fever virus (ASFV), can reach 100%. ASF has been reported in 25 Chinese provinces 15 since August 2018. There is no effective treatment or vaccine for it and the present molecular diagnosis 16 technologies have trade-offs in sensitivity, specificity, cost and speed, and none of them cater perfectly 17 to ASF control. Thus, a technology that overcomes the need for laboratory facilities, i… Show more
“…In most of these platforms, a commercially available universal test strip, the HybriDetect-Universal Lateral Flow Assay Kit, was used. This dipstick was originally designed for qualitative or even quantitative rapid testing of proteins, antibodies or gene amplicons, but has been adapted to function in several LFA based CRISPR sensing methods ( Bai et al, 2019 ; Chang et al, 2019 ; Gootenberg et al, 2018 ; Kaminski et al, 2020 ; Mukama et al, 2020b ; Sullivan et al, 2019 ; Tsou et al, 2019 ). This platform offers a Streptavidine line as well as an antibody line that can capture anti-FITC coated AuNPs ( Fig.…”
Section: Crispr/cas Sensing In Poc Sensorsmentioning
confidence: 99%
“… Fig. 4 A) Type V/VI based LFA, adapted from ( Bai et al, 2019 ) with permission from Creative Commons Attribution License (CC BY) B) Type II based LFA, adapted from with permission from ( Wang et al, 2020 ). Copyright 2020 American Chemical Society.…”
Section: Crispr/cas Sensing In Poc Sensorsmentioning
With the trend of moving molecular tests from clinical laboratories to on-site testing, there is a need for nucleic acid based diagnostic tools combining the sensitivity, specificity and flexibility of established diagnostics with the ease, cost effectiveness and speed of isothermal amplification and detection methods. A promising new nucleic acid detection method is Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated nuclease (Cas)-based sensing. In this method Cas effector proteins are used as highly specific sequence recognition elements that can be combined with many different read-out methods for on-site point-of-care testing. This review covers the technical aspects of integrating CRISPR/Cas technology in miniaturized sensors for analysis on-site. We start with a short introduction to CRISPR/Cas systems and the different effector proteins and continue with reviewing the recent developments of integrating CRISPR sensing in miniaturized sensors for point-of-care applications. Finally, we discuss the challenges of point-of-care CRISPR sensing and describe future research perspectives.
“…In most of these platforms, a commercially available universal test strip, the HybriDetect-Universal Lateral Flow Assay Kit, was used. This dipstick was originally designed for qualitative or even quantitative rapid testing of proteins, antibodies or gene amplicons, but has been adapted to function in several LFA based CRISPR sensing methods ( Bai et al, 2019 ; Chang et al, 2019 ; Gootenberg et al, 2018 ; Kaminski et al, 2020 ; Mukama et al, 2020b ; Sullivan et al, 2019 ; Tsou et al, 2019 ). This platform offers a Streptavidine line as well as an antibody line that can capture anti-FITC coated AuNPs ( Fig.…”
Section: Crispr/cas Sensing In Poc Sensorsmentioning
confidence: 99%
“… Fig. 4 A) Type V/VI based LFA, adapted from ( Bai et al, 2019 ) with permission from Creative Commons Attribution License (CC BY) B) Type II based LFA, adapted from with permission from ( Wang et al, 2020 ). Copyright 2020 American Chemical Society.…”
Section: Crispr/cas Sensing In Poc Sensorsmentioning
With the trend of moving molecular tests from clinical laboratories to on-site testing, there is a need for nucleic acid based diagnostic tools combining the sensitivity, specificity and flexibility of established diagnostics with the ease, cost effectiveness and speed of isothermal amplification and detection methods. A promising new nucleic acid detection method is Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated nuclease (Cas)-based sensing. In this method Cas effector proteins are used as highly specific sequence recognition elements that can be combined with many different read-out methods for on-site point-of-care testing. This review covers the technical aspects of integrating CRISPR/Cas technology in miniaturized sensors for analysis on-site. We start with a short introduction to CRISPR/Cas systems and the different effector proteins and continue with reviewing the recent developments of integrating CRISPR sensing in miniaturized sensors for point-of-care applications. Finally, we discuss the challenges of point-of-care CRISPR sensing and describe future research perspectives.
The mortality rate of hemorrhagic African swine fever (ASF), which targets domestic pigs and wild boars is caused by African swine fever virus (ASFV), can reach 100%. Since the first confirmed ASF outbreak in China on 3 August 2018, 156 ASF outbreaks were detected in 32 provinces. About 1,170,000 pigs were culled in order to halt further spread. There is no effective treatment or vaccine for it and the present molecular diagnosis technologies have trade-offs in sensitivity, specificity, cost and speed, and none of them cater perfectly to ASF control. Thus, a technology that overcomes the need for laboratory facilities, is relatively low cost, and rapidly and sensitively detects ASFV would be highly valuable. Here, we describe an RAA-Cas12a-based system that combines recombinase aided amplification (RAA) and CRISPR/Cas12a for ASFV detection. The fluorescence intensity readout of this system detected ASFV p72 gene levels as low as 10 aM. For on-site ASFV detection, lateral-flow strip readout was introduced for the first time in the RAA-Cas12a based system (named CORDS, Cas12abased On-site and Rapid Detection System). We used CORDS to detect target DNA highly specifically using the lateral-flow strip readout and the assay displayed no crossreactivity to other 13 swine viruses including classical swine fever (CSF). CORDS could identify the ASFV DNA target at femtomolar sensitivity in an hour at 37 • C, and only requires an incubator. For ease of use, the reagents of CORDS were lyophilized to three tubes and remained the same sensitivity when stored at 4 • C for at least 7 days. Thus, CORDS provide a rapid, sensitive and easily operable method for ASFV on-site detection. Lyophilized CORDS can withstand long-term transportation and storage, and is ready for field-based applications.
“…Importantly, the simplicity of the CRISPR/Cas-based detection system allows the rapid development of diagnostic methods for an emergency outbreak of infectious diseases. For example, integrating later flow assay (CORDS, CRISPR/Cas12a-LFD) and naked-eye detection (CRISPR/Cas-based colorimetric platform, fluorescence-based POC system), four groups quickly developed Cas12a-based on-site diagnostic assays for African swine virus (ASFV), an emergence virus for the global swine industry (Table 2) [44][45][46][47]. For the current COVID-19 pandemic [48], several research groups are developing CRISPR/Cas-based diagnostic assay [49][50][51][52][53].…”
Section: The Strengths Of Crispr/cas Based Pathogen Detection Systemsmentioning
Ideal methods for detecting pathogens should be sensitive, specific, rapid, cost-effective and instrument-free. Conventional nucleic acid pathogen detection strategies, mostly PCR-based techniques, have various limitations, such as expensive equipment, reagents and skilled performance. Recently, CRISPR/Cas-based methods have burst onto the scene, with the potential to power the pathogen detection field. Here we introduce these unique methods and discuss its hurdles and promises.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
