Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 Triggered Isothermal Amplification for Site-Specific Nucleic Acid Detection

Huang, Mingzhi; Zhou, Xiaoming; Wang, Huiying; Xing, Da

doi:10.1021/acs.analchem.7b04542

Cited by 218 publications

(172 citation statements)

References 51 publications

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“…With its ability to accurately recognize specific sequence, the CRISPR/Cas system holds promise as a detection method (Huang et al, 2018;Sashital, 2018;Li et al, 2019). The transcleavage activity of the recently reported Cas12a, Cas13a, and Cas14 systems in particular, helps them first bind to the target specifically and then amplify the binding signal by cutting the dual-labeled probe in a nonspecific way (Gootenberg et al, 2017;Chen et al, 2018;Harrington et al, 2018;Qian et al, 2019).…”

Section: Introductionmentioning

confidence: 99%

Cas12a-Based On-Site and Rapid Nucleic Acid Detection of African Swine Fever

et al. 2019

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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.

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Section: Introductionmentioning

confidence: 99%

Cas12a-Based On-Site and Rapid Nucleic Acid Detection of African Swine Fever

et al. 2019

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“…In the recent past, studies, employing CRISPR‐associated methods for the detection of nucleic acids, have been utilized using different CRISPR‐associated (Cas) effectors . Varying Cas effectors were used for targeting different nucleic acids, like the Cas9 effector for the detection of double‐stranded DNA (dsDNA), along with the Cas12 for the detection of single‐stranded DNA (ssDNA) . The microbial CRISPR effector Cas13a (previously named C2c2), displays, in contrast to the other Cas effectors, a triggered cleavage capability of nontarget single‐stranded RNAs (ssRNAs) in the surrounding .…”

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confidence: 99%

CRISPR/Cas13a‐Powered Electrochemical Microfluidic Biosensor for Nucleic Acid Amplification‐Free miRNA Diagnostics

et al. 2019

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Noncoding small RNAs, such as microRNAs, are becoming the biomarkers of choice for multiple diseases in clinical diagnostics. A dysregulation of these microRNAs can be associated with many different diseases, such as cancer, dementia, and cardiovascular conditions. The key for effective treatment is an accurate initial diagnosis at an early stage, improving the patient's survival chances. In this work, the first clustered regularly interspaced short palindromic repeats (CRISPR)/Cas13a‐powered microfluidic, integrated electrochemical biosensor for the on‐site detection of microRNAs is introduced. Through this unique combination, the quantification of the potential tumor markers microRNA miR‐19b and miR‐20a is realized without any nucleic acid amplification. With a readout time of 9 min and an overall process time of less than 4 h, a limit of detection of 10 pm is achieved, using a measuring volume of less than 0.6 µL. Furthermore, the feasibility of the biosensor platform to detect miR‐19b in serum samples of children, suffering from brain cancer, is demonstrated. The validation of the obtained results with a standard quantitative real‐time polymerase chain reaction method shows the ability of the electrochemical CRISPR‐powered system to be a low‐cost, easily scalable, and target amplification‐free tool for nucleic acid based diagnostics.

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“…In 2018, a Cas9-based nucleotide detection tool termed CAS-EXPAR (Cas9 triggered exponential amplification reaction) was developed. Combined with exponential amplification reaction, CAS-EXPAR could detect the target at concentration as low as 0.82 amol (0.82 × 10 −18 mol/L) (Huang et al, 2018). Subsequently, this technology was optimized to monitor methylation status of DNA fragment.…”

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confidence: 99%