Digital CRISPR/Cas12b-based platform enabled absolute quantification of viral RNA

Luo, Xinyi; Xue, Yingying; Ju, Enguo; Yu, Tao; Li, Mingqiang; Zhou, Li; Yang, Chongguang; Zhou, Jianhua; Wang, Jiasi

doi:10.1016/j.aca.2021.339336

Cited by 34 publications

(29 citation statements)

References 30 publications

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“…The CRISPR/Cas systems including a guide RNA (gRNA) and a Cas12a protein are widely utilized to improve the sensitivity and specificity of molecular assays [ 71 ]. They make the determination of nucleic acids possible with a single molecular sensitivity [ 72 ]. So, the CRISPR/Cas systems can play an important role in the global fight against the COVID-19 pandemic using the SARS-CoV-2 detection [ 73 ].…”

Section: Colorimetric Sensing Methodsmentioning

confidence: 99%

AuNP-based biosensors for the diagnosis of pathogenic human coronaviruses: COVID-19 pandemic developments

2022

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The outbreak rate of human coronaviruses (CoVs) especially highly pathogenic CoVs is increasing alarmingly. Early detection of these viruses allows treatment interventions to be provided more quickly to people at higher risk, as well as helping to identify asymptomatic carriers and isolate them as quickly as possible, thus preventing the disease transmission chain. The current diagnostic methods such as RT-PCR are not ideal due to high cost, low accuracy, low speed, and probability of false results. Therefore, a reliable and accurate method for the detection of CoVs in biofluids can become a front-line tool in order to deal with the spread of these deadly viruses. Currently, the nanomaterial-based sensing devices for detection of human coronaviruses from laboratory diagnosis to point-of-care (PoC) diagnosis are progressing rapidly. Gold nanoparticles (AuNPs) have revolutionized the field of biosensors because of the outstanding optical and electrochemical properties. In this review paper, a detailed overview of AuNP-based biosensing strategies with the varied transducers (electrochemical, optical, etc.) and also different biomarkers (protein antigens and nucleic acids) was presented for the detection of human coronaviruses including SARS-CoV-2, SARS-CoV-1, and MERS-CoV and lowly pathogenic CoVs. The present review highlights the newest trends in the SARS-CoV-2 nanobiosensors from the beginning of the COVID-19 epidemic until 2022. We hope that the presented examples in this review paper convince readers that AuNPs are a suitable platform for the designing of biosensors. Graphical abstract

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Section: Colorimetric Sensing Methodsmentioning

confidence: 99%

AuNP-based biosensors for the diagnosis of pathogenic human coronaviruses: COVID-19 pandemic developments

2022

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“…Numerous CRISPR-based nucleic acid-sensing systems were reported in the past several years. ,, The FOM model described in eq provides us with a tool to benchmark the performance of these different systems. We studied a total of 57 published works (Table ) related to CRISPR-based nucleic acid sensing up to this date (Feb 2022). ,,− ,− ,− ,,,,,,,,,,,,,− It is noteworthy that while many more CRISPR-based sensing studies have been published in the past few years, we only include those with the LOD and CRISPR reaction time available. It should be mentioned that Cas9 , and Cas14 have been utilized for diagnostics.…”

Section: Performance Benchmarkingmentioning

confidence: 99%

Figure of Merit for CRISPR-Based Nucleic Acid-Sensing Systems: Improvement Strategies and Performance Comparison

et al. 2022

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Clustered regularly interspaced short palindromic repeats (CRISPR)-based nucleic acid-sensing systems have grown rapidly in the past few years. Nevertheless, an objective approach to benchmark the performances of different CRISPR sensing systems is lacking due to the heterogeneous experimental setup. Here, we developed a quantitative CRISPR sensing figure of merit (FOM) to compare different CRISPR methods and explore performance improvement strategies. The CRISPR sensing FOM is defined as the product of the limit of detection (LOD) and the associated CRISPR reaction time (T). A smaller FOM means that the method can detect smaller target quantities faster. We found that there is a tradeoff between the LOD of the assay and the required reaction time. With the proposed CRISPR sensing FOM, we evaluated five strategies to improve the CRISPR-based sensing: preamplification, enzymes of higher catalytic efficiency, multiple crRNAs, digitalization, and sensitive readout systems. We benchmarked the FOM performances of 57 existing studies and found that the effectiveness of these strategies on improving the FOM is consistent with the model prediction. In particular, we found that digitalization is the most promising amplification-free method for achieving comparable FOM performances (∼1 fM·min) as those using preamplification. The findings here would have broad implications for further optimization of the CRISPR-based sensing.

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“…However, the popular isothermal amplification method LAMP requires higher temperatures (60–65 °C) than RPA (37–42 °C) and is therefore incompatible with the operational temperatures of Cas12a and Cas13a. A drop study therefore used Cas12b, which has greater thermal stability, to demonstrate absolute quantification of SARS-CoV-2 RNA in conjunction with RT-LAMP …”

Section: Viral Detection and Quantificationmentioning

confidence: 99%

Droplet Microfluidics for High-Resolution Virology

Jing

Han

2022

Anal. Chem.

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Microfluidics has enabled a new era of cellular and molecular assays due to the small length scales, parallelization, and the modularity of various analysis and actuation functions. Droplet microfluidics, in particular, has been instrumental in providing new tools for biology with its ability to quickly and reproducibly generate drops that act as individual reactors. A notable beneficiary of this technology has been single-cell RNA sequencing, which has revealed new heterogeneities and interactions for the fundamental unit of life. However, viruses far surpass the diversity of cellular life, affect the dynamics of all ecosystems, and are a chronic source of global health crises. Despite their impact on the world, high-throughput and high-resolution viral profiling has been difficult, with conventional methods being limited to population-level averaging, large sample volumes, and few cultivable hosts. Consequently, most viruses have not been identified and studied. Droplet microfluidics holds the potential to address many of these limitations and offers new levels of sensitivity and throughput for virology. This Feature highlights recent efforts that have applied droplet microfluidics to the detection and study of viruses, including for diagnostics, virus−host interactions, and cell-independent virus assays. In combination with traditional virology methods, droplet microfluidics should prove a potent tool toward achieving a better understanding of the most abundant biological species on Earth.

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Digital CRISPR/Cas12b-based platform enabled absolute quantification of viral RNA

Cited by 34 publications

References 30 publications

AuNP-based biosensors for the diagnosis of pathogenic human coronaviruses: COVID-19 pandemic developments

AuNP-based biosensors for the diagnosis of pathogenic human coronaviruses: COVID-19 pandemic developments

Figure of Merit for CRISPR-Based Nucleic Acid-Sensing Systems: Improvement Strategies and Performance Comparison

Droplet Microfluidics for High-Resolution Virology

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