A one-step, one-pot CRISPR nucleic acid detection platform (CRISPR-top): Application for the diagnosis of COVID-19

Li, Shijun; Huang, Junfei; Ren, Lijuan; Jiang, Weijia; Wang, Ming; Li, Zhuang; Zheng, Qinni; Yang, Rui; Zeng, Yi; Luu, Laurence Don Wai; Wang, Yi; Tai, Jun

doi:10.1016/j.talanta.2021.122591

Cited by 58 publications

(60 citation statements)

References 22 publications

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“…Also, RT-RPA can be run in parallel (one-pot test) with CRISPR-Cas systems from mesophilic microorganisms. For RT-LAMP, the only way to circumvent the temperature limitation and to develop one-pot methods is using temperature-resistant EnAs-Cas12a (works at 60 ºC) or thermostable AapCas12b, which run simultaneously with the RT-LAMP at 59-62 ºC [26,40,51,52].…”

Section: Time and Temperature Of Isothermal Methodsmentioning

confidence: 99%

“…The most common types are nasopharyngeal (NP) and/or oropharyngeal (OP) swabs. Likewise, saliva and sputum samples (15% of the analyzed methods) [24][25][26][27][28][29][30], which allow self-sampling directly by the patients [31][32][33], have been used successfully to detect the virus. Other less used types of samples include bronchoalveolar lavage fluid, anal swabs, stool [26,27] and harvested lentivirus samples [34].…”

Section: Type Of Samplementioning

confidence: 99%

“…Not many details about the costs of the methods have been reported. Indeed, we just found 6 methods that reported a cost estimation [20,26,35,37,52,56]. The costs range from 1 to 10 USD per reaction (usually only including the cost of the proteins, gRNAs, and other materials).…”

Section: Cost and Manufacturingmentioning

confidence: 99%

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Diagnostics of COVID-19 based on CRISPR-Cas coupled to Isothermal Amplification: A Comparative Analysis and Update

Hernández-García¹,

Morales²,

Galindo³

et al. 2022

Preprint

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The emergence of the COVID-19 pandemics prompted a fast development of novel diagnostic methods of the etiologic virus SARS-CoV-2. Methods based on CRISPR-Cas systems have been particularly promising because they can achieve a similar sensitivity and specificity to the golden standard RT-qPCR, especially when coupled to an isothermal pre-amplification step. Furthermore, they have also solved inherent limitations of RT-qPCR that impede its decentralized use and deployment in the field, such as the need for expensive equipment, high cost per reaction, and delivery of results in hours, among others. In this review, we evaluate publicly available methods to detect SARS-CoV-2 that are based on CRISPR-Cas and isothermal amplification. We critically analyze the steps required to obtain a successful result from clinical samples and pinpoint key experimental conditions and parameters that could be optimized or modified to improve clinical and analytical outputs. The COVID outbreak has propelled intensive research in a short time, which is paving the way to develop effective and very promising CRISPR-Cas systems for the precise detection of SARS-CoV-2. This review could also serve as an introductory guide to new labs delving into this technology.

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Section: Time and Temperature Of Isothermal Methodsmentioning

confidence: 99%

Section: Type Of Samplementioning

confidence: 99%

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Diagnostics of COVID-19 based on CRISPR-Cas coupled to Isothermal Amplification: A Comparative Analysis and Update

Hernández-García¹,

Morales²,

Galindo³

et al. 2022

Preprint

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“…A pair of PCR primers to amplify a 174 bp sequence in the gyrA gene was designed using Primer Premier 6.0. Then, an ATTTA sequence was added on the 5′ end of the forward primer as the PAM for CRISPR-Cas12a-based detection (Li et al, 2021). Moreover, an efficient CRISPR RNA (crRNA) was designed according to the amplification site (Figure 2 and Table S2).…”

Section: Primers and Crrna Designmentioning

confidence: 99%

A CRISPR-based nucleic acid detection platform (CRISPR-CPA): Application for detection of Nocardia farcinica

Qiu

Liu

et al. 2022

Journal of Applied Microbiology

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Aims To establish a CRISPR‐based nucleic acid detection platform and apply it to the detection of Nocardia farcinica. Methods and Results A CRISPR‐based nucleic acid detection platform, termed CRISPR‐CPA (CRISPR/Cas12a combined with PCR amplification), which employed PCR for pre‐amplification of target sequences and CRISPR‐Cas12a‐based detection for decoding of the PCR amplicons, was developed. To demonstrate its feasibility, CRISPR‐CPA was applied to the detection of N. farcinica. A pair of PCR primers and a crRNA, which targeting the conservative and specific part of gyrA of N. farcinica reference strain IFM 10152, were designed according to the principle of CRISPR‐CPA. The whole detection process of N. farcinica CRISPR‐CPA assay, including sample pre‐treatment and DNA extraction (~20 min), PCR pre‐amplification (60 min), CRISPR‐based detection (10 min), can be completed within 90 min. A total of 62 isolates were used to evaluate the specificity of N. farcinica CRISPR‐CPA assay. Clinical specimens were employed to determine the feasibility of the method in practical application. The limit of detection of the N. farcinica CRISPR‐CPA assay is 1 pg DNA per reaction in pure cultures and 105 CFU/ml in sputum specimens, which is similar with culture but significantly more timesaving. Conclusions The N. farcinica CRISPR‐CPA assay is an economic and specific method to detect N. farcinica and provides a high‐efficiency tool for screening of pathogens especially of some hard‐to‐culture and slow‐growth infectious agents. Significance and Impact of the Study In CRISPR‐CPA system, the PCR primers are engineered with a protospacer adjacent motif (PAM) site of Cas12a effector and an additional base A was added at the 5′ end of the engineered PCR primer for protecting PAM site, thus the CRISPR‐CPA can detect any sequence. Also, we applied CRISPR‐CPA to rapidly detect N. farcinica, which is slow‐growing bacteria and is firstly detected by a CRISPR‐based method.

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“…Nucleic acid detection is currently the gold standard for detecting SARS-CoV-2, and the primary detection tool with the advantages of early diagnosis, high sensitivity, and specificity [ 8 , 9 ]. Current nucleic acid-based assays include complete genome sequence [ 10 , 11 ], real-time polymerase chain reaction (RT-PCR) [ 12 , 13 ], isothermal nucleic acid amplification [ 14 , 15 ], CRISPR [ 16 , 17 ], enzyme-linked immunosorbent assay [ 18 ], infrared absorption spectroscopy [ 19 ], and biosensor detection [ 20 , 21 , 22 ]. As the probability of genetic mutation and complexity of SARS-CoV-2 strains increase during the spreading process [ 23 ] despite rapid detection through existing methods, there is increased threat to human life and health.…”

Section: Introductionmentioning

confidence: 99%

A Study of the Detection of SARS-CoV-2 ORF1ab Gene by the Use of Electrochemiluminescent Biosensor Based on Dual-Probe Hybridization

Jiang¹,

Mu²,

Liu³

et al. 2022

Sensors

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To satisfy the need to develop highly sensitive methods for detecting the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) and further enhance detection efficiency and capability, a new method was created for detecting SARS-CoV-2 of the open reading frames 1ab (ORF1ab) target gene by a electrochemiluminescence (ECL) biosensor based on dual-probe hybridization through the use of a detection model of “magnetic capture probes—targeted nucleic acids—Ru(bpy)32+ labeled signal probes”. The detection model used magnetic particles coupled with a biotin-labeled complementary nucleic acid sequence of the SARS-CoV-2 ORF1ab target gene as the magnetic capture probes and Ru(bpy)32+ labeled amino modified another complementary nucleic acid sequence as the signal probes, which combined the advantages of the highly specific dual-probe hybridization and highly sensitive ECL biosensor technology. In the range of 0.1 fM~10 µM, the method made possible rapid and sensitive detection of the ORF1ab gene of the SARS-CoV-2 within 30 min, and the limit of detection (LOD) was 0.1 fM. The method can also meet the analytical requirements for simulated samples such as saliva and urine with the definite advantages of a simple operation without nucleic acid amplification, high sensitivity, reasonable reproducibility, and anti-interference solid abilities, expounding a new way for efficient and sensitive detection of SARS-CoV-2.

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A one-step, one-pot CRISPR nucleic acid detection platform (CRISPR-top): Application for the diagnosis of COVID-19

Cited by 58 publications

References 22 publications

Diagnostics of COVID-19 based on CRISPR-Cas coupled to Isothermal Amplification: A Comparative Analysis and Update

Diagnostics of COVID-19 based on CRISPR-Cas coupled to Isothermal Amplification: A Comparative Analysis and Update

A CRISPR-based nucleic acid detection platform (CRISPR-CPA): Application for detection of Nocardia farcinica

A Study of the Detection of SARS-CoV-2 ORF1ab Gene by the Use of Electrochemiluminescent Biosensor Based on Dual-Probe Hybridization

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