Rotational scan digital LAMP for accurate quantitation of nucleic acids

Jiang, Mi; Liao, Peiyu; Sun, Yue; Shao, Xinyang; Chen, Zitian; Fei, Peng; Wang, Jianbin; Huang, Yanyi

doi:10.1039/d1lc00114k

Cited by 7 publications

(3 citation statements)

References 41 publications

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“…It was shown that a large amount (>10 000) of monodisperse, water-in-oil microdroplets could be generated upon high-speed centrifugation 37 (Figure 2). In addition, microdroplets could be made in parallel using many pipette-tip microdroplet generators when centrifugation was applied to the system (Figure 2a,b).…”

Section: ■ Results and Discussionmentioning

confidence: 99%

Pipette-Tip-Enabled Digital Nucleic Acid Analyzer for COVID-19 Testing with Isothermal Amplification

et al. 2021

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Herein, a pipette-tip-enabled digital nucleic acid analyzer for high-performance COVID-19 testing is demonstrated. This is achieved by digital loop-mediated isothermal amplification (digital LAMP or dLAMP) using common laboratory equipment and materials. It is shown that simply fixing a glass capillary inside conventional pipette tips enables the generation of monodisperse, water-in-oil microdroplets with benchtop centrifugation. It is shown that using LAMP, the ORF1a/b gene, a standard test region for COVID-19 screening, can be amplified without a thermal cycler. The amplification allows counting of fluorescent microdroplets so that Poisson analysis can be performed to allow quantification with a limit of detection that is 1 order of magnitude better than those of nondigital techniques and comparable to those of commercial dLAMP platforms. It is envisioned that this work will inspire studies on ultrasensitive digital nucleic acid analyzers demanding both sensitivity and accessibility, which is pivotal to their large-scale applications.

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Section: ■ Results and Discussionmentioning

confidence: 99%

Pipette-Tip-Enabled Digital Nucleic Acid Analyzer for COVID-19 Testing with Isothermal Amplification

et al. 2021

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“…Based on that, a linear range of 25–10,000 copy μL –1 in the LAMP mixture with an RSD of 6.3% ( n = 7, c = 100 copy μL –1 ) was obtained. A comparison of this work with other works of digital assays ,,− or element-labeling ICP-MS and spICP-MS assays ,− for nucleic acids is listed in Table S3. The lower limit of the linear range of this work is higher than most of the works of digital assay due to the relatively low transport efficiency of 2.6% of ICP-MS.…”

Section: Resultsmentioning

confidence: 99%

Agarose-Droplet-Based Digital LAMP Assay for Counting Virus DNA in Single-Particle ICP-MS

Xiao

Chen

et al. 2022

Anal. Chem.

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Inductively coupled plasma mass spectrometry (ICP-MS) has emerged as a promising analytical platform for the quantification of biomolecules using elemental tags; however, absolute quantification at extremely low concentrations by ICP-MS without a calibration curve remains challenging. Here, we developed a digital loop-mediated isothermal amplification (LAMP) assay for counting hepatitis B virus (HBV) DNA using single-particle (sp) ICP-MS. The sample and LAMP reagents were mixed and encapsulated in agarose droplets, which were generated by homemade centrifugal droplet generators. The agarose droplets were incubated at 65 °C for amplifying the virus DNA with LAMP primers and then cooled to 4 °C for generating “gel” particles during the temperature-dependent “sol–gel” transition. The LAMP amplicons were intercalated into the agarose particles using polyacrylamide-modified LAMP primers, enabling the labeling of dsDNA with [Ru(bpy)2dppz]2+ and the removal of excess reagents. Only those agarose particles, containing virus DNA, could be labeled with 101Ru and detected in spICP-MS. We also embedded the 153Eu-containing polystyrene microspheres into agarose droplets as the internal standard for counting the total number of agarose droplets. The copy number of virus DNA could be counted from the 101Ru/153Eu pulse numbers in spICP-MS. We achieved the lowest quantification of 25 copy μL–1 virus DNA in one analysis without the need for a calibration curve. The developed assay can be easily tuned for counting multiple types of nucleic acid targets and extended for new possibilities of the spICP-MS-based digital assay.

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“…To further apply MiCA to d-PCR assay, specific aqueous and oil phases were formulated to ensure stable thermal and mechanical properties of the generated droplets [18]. The latest developments of this technique include the refractive indices matching of MiCA emulsion and the combination with advanced 3D fluorescence microscopy or tomography techniques [19][20][21][22][23][24][25][26][27] to together offer lossless and contamination-free droplet generation as well as high-speed, in-parallel signal readout in a single PCR tube [28,29] The most state-of-the-art development, term CLEAR d-PCR has shown high performances, such as speed, dynamic range, and accuracy, superior to other alternative d-PCR technologies. However, a major weakness of current CLEAR d-PCR is the use of a conventional selective plane illumination microscopy (SPIM) setup, which is optimized for imaging a single centrifuge tube at one time.…”

Section: Introductionmentioning

confidence: 99%

Open-top light-sheet imaging of CLEAR emulsion for high-throughput loss-free analysis of massive fluorescent droplets

Jiang¹,

Chen²,

Wang³

et al. 2022

Biomed. Phys. Eng. Express

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Digital droplet PCR (ddPCR) is classified as the third-generation PCR technology that enables absolute quantitative detection of nucleic acid molecules and has become an increasingly powerful tool for clinic diagnosis. We previously established a CLEAR-dPCR technique based on the combination of CLEAR droplets generated by micro-centrifuge-based microtubule arrays (MiCA) and insitu 3D readout by light-sheet fluorescence imaging. This CLEAR-dPCR technique attains very high readout speed and dynamic range. Meanwhile, it is free from sample loss and contamination, showing its advantages over commercial d-PCR technologies. However, a conventional orthogonal light-sheet imaging setup in CLEAR d-PCR cannot image multiple centrifuge tubes, thereby limiting its widespread application to large-scale, high-speed dd-PCR assays. Herein, we propose an in-parallel 3D dd-PCR readout technique based on an open-top light-sheet microscopy setup. This approach can continuously scan multiple centrifuge tubes which contain CLEAR emulsions with highly diverse concentrations, and thus further boost the scale and throughput of our 3D dd-PCR technique.

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Rotational scan digital LAMP for accurate quantitation of nucleic acids

Abstract: Digital quantitation of nucleic acids is precise and sensitive because of its molecular-level resolution. However, only several quantitation formats are common, especially pertaining to how one obtains digital signals from...

Cited by 7 publications

References 41 publications

Pipette-Tip-Enabled Digital Nucleic Acid Analyzer for COVID-19 Testing with Isothermal Amplification

Pipette-Tip-Enabled Digital Nucleic Acid Analyzer for COVID-19 Testing with Isothermal Amplification

Agarose-Droplet-Based Digital LAMP Assay for Counting Virus DNA in Single-Particle ICP-MS

Open-top light-sheet imaging of CLEAR emulsion for high-throughput loss-free analysis of massive fluorescent droplets

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