Reverse Transcription Recombinase Polymerase Amplification Coupled with CRISPR-Cas12a for Facile and Highly Sensitive Colorimetric SARS-CoV-2 Detection

Zhang, Wei S.; Pan, Jianbin; Feng, Li; Zhu, Min; Xu, Mengting; Zhu, Hongyan; Yu, Yanyan; Su, Gaoxing

doi:10.1021/acs.analchem.1c00013

Cited by 207 publications

(137 citation statements)

References 50 publications

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“…More importantly, in combination with lateral flow test strip technology, the results can be visible to the naked eye, and have great practical value in home testing. Fluorescence readout can be achieved with portable fluorescence collection instruments, or with point-of-care detection by colorimetric analysis [ 25 , 26 , 27 ]. In the present study, the LoD of LAMP-Cas12a was 1 copy/μL.…”

Section: Discussionmentioning

confidence: 99%

CRISPR/Cas12-Based Ultra-Sensitive and Specific Point-of-Care Detection of HBV

Ding

Long

Yuan

et al. 2021

IJMS

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Hepatitis B remains a major global public health challenge, with particularly high prevalence in medically disadvantaged western Pacific and African regions. Although clinically available technologies for the qPCR detection of HBV are well established, research on point-of-care testing has not progressed substantially. The development of a rapid, accurate point-of-care test is essential for the prevention and control of hepatitis B in medically disadvantaged rural areas. The development of the CRISPR/Cas system in nucleic acid detection has allowed for pathogen point-of-care detection. Here, we developed a rapid and accurate point-of-care assay for HBV based on LAMP-Cas12a. It innovatively solves the problem of point-of-care testing in 10 min, particularly the problem of sample nucleic acid extraction. Based on LAMP-Cas12a, visualization of the assay results is presented by both a fluorescent readout and by lateral flow test strips. The lateral flow test strip technology can achieve results visible to the naked eye, while fluorescence readout can achieve real-time high-sensitivity detection. The fluorescent readout-based Cas12a assay can achieve HBV detection with a limit of detection of 1 copy/μL within 13 min, while the lateral flow test strip technique only takes 20 min. In the evaluation of 73 clinical samples, the sensitivity and specificity of both the fluorescence readout and lateral flow test strip method were 100%, and the results of the assay were fully comparable to qPCR. The LAMP-Cas12a-based HBV assay relies on minimal equipment to provide rapid, accurate test results and low costs, providing significant practical value for point-of-care HBV detection.

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Section: Discussionmentioning

confidence: 99%

CRISPR/Cas12-Based Ultra-Sensitive and Specific Point-of-Care Detection of HBV

Ding

Long

Yuan

et al. 2021

IJMS

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“…The L/RPA assay showed a sensitivity of 10 1 viral RNA copies per reaction, which was comparable to that of RT-qPCR and other nucleic acid detection assays for SARS-CoV-2 (Behrmann et al, 2020;Zhang C. et al, 2021;Zhang W. S. et al, 2021). The assay could be finished in less than 30 min with simple operation and without dependence on sophisticated thermocycling equipment.…”

Section: Discussionmentioning

confidence: 81%

“…Rapid and simple SARS-CoV-2 detection methods that are not limited by sophisticated thermocycling equipment are valuable for diagnosis needs in resource-limited areas, family self-testing or mass screening. For this purpose, assays based on LAMP, RPA and CRISPR technologies have been developed (Rabe and Cepko, 2020;Haq et al, 2021;Zhang W. S. et al, 2021). These assays are generally faster and simpler than RT-qPCR, providing more choices for the testing needs of SARS-CoV-2 under different situations.…”

Section: Discussionmentioning

confidence: 99%

A Ligation/Recombinase Polymerase Amplification Assay for Rapid Detection of SARS-CoV−2

Wang

Ma²,

Zhang³

et al. 2021

Front. Cell. Infect. Microbiol.

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The pandemic of COVID-19 caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has led to more than 117 million reported cases and 2.6 million deaths. Accurate diagnosis technologies are vital for controlling this pandemic. Reverse transcription (RT)-based nucleic acid detection assays have been developed, but the strict sample processing requirement of RT has posed obstacles on wider applications. This study established a ligation and recombinase polymerase amplification (L/RPA) combined assay for rapid detection of SARS-CoV−2 on genes N and ORF1ab targeting the specific biomarkers recommended by the China CDC. Ligase-based strategies usually have a low-efficiency problem on RNA templates. This study has addressed this problem by using a high concentration of the T4 DNA ligase and exploiting the high sensitivity of RPA. Through selection of the ligation probes and optimization of the RPA primers, the assay achieved a satisfactory sensitivity of 101 viral RNA copies per reaction, which was comparable to RT-quantitative polymerase chain reaction (RT-qPCR) and other nucleic acid detection assays for SARS-CoV−2. The assay could be finished in less than 30 min with a simple procedure, in which the requirement for sophisticated thermocycling equipment had been avoided. In addition, it avoided the RT procedure and could potentially ease the requirement for sample processing. Once validated with clinical samples, the L/RPA assay would increase the practical testing availability of SARS-CoV-2. Moreover, the principle of L/RPA has an application potential to the identification of concerned mutations of the virus.

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“…For SARS-CoV-2 detection, researchers have optimized RPA procedures by testing various experimental parameters that can now detect less than five viral copies from patient samples within 45 min from the sample collection [ 134 ]. RPA methods have also been optimized for SARS-CoV-2 detection by coupling RPA-based amplification with various CRISPR-based detection methods [ 155 , 156 , 157 ].…”

Section: Isothermal Nucleic Acid Amplification Methodsmentioning

confidence: 99%

Nucleic Acid Testing of SARS-CoV-2

Yoo

Kim

2021

IJMS

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The coronavirus disease 2019 (COVID-19) has caused a large global outbreak. It is accordingly important to develop accurate and rapid diagnostic methods. The polymerase chain reaction (PCR)-based method including reverse transcription-polymerase chain reaction (RT-PCR) is the most widely used assay for the detection of SARS-CoV-2 RNA. Along with the RT-PCR method, digital PCR has emerged as a powerful tool to quantify nucleic acid of the virus with high accuracy and sensitivity. Non-PCR based techniques such as reverse transcription loop-mediated isothermal amplification (RT-LAMP) and reverse transcription recombinase polymerase amplification (RT-RPA) are considered to be rapid and simple nucleic acid detection methods and were reviewed in this paper. Non-conventional molecular diagnostic methods including next-generation sequencing (NGS), CRISPR-based assays and nanotechnology are improving the accuracy and sensitivity of COVID-19 diagnosis. In this review, we also focus on standardization of SARS-CoV-2 nucleic acid testing and the activity of the National Metrology Institutes (NMIs) and highlight resources such as reference materials (RM) that provide the values of specified properties. Finally, we summarize the useful resources for convenient COVID-19 molecular diagnostics.

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Reverse Transcription Recombinase Polymerase Amplification Coupled with CRISPR-Cas12a for Facile and Highly Sensitive Colorimetric SARS-CoV-2 Detection

Cited by 207 publications

References 50 publications

CRISPR/Cas12-Based Ultra-Sensitive and Specific Point-of-Care Detection of HBV

CRISPR/Cas12-Based Ultra-Sensitive and Specific Point-of-Care Detection of HBV

A Ligation/Recombinase Polymerase Amplification Assay for Rapid Detection of SARS-CoV−2

Nucleic Acid Testing of SARS-CoV-2

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