Multiplexed droplet loop-mediated isothermal amplification with scorpion-shaped probes and fluorescence microscopic counting for digital quantification of virus RNAs

Tan, Ya-Ling; Huang, A-Qian; Tang, Li‐Juan; Jiang, Jian‐Hui

doi:10.1039/d1sc00616a

Cited by 28 publications

(22 citation statements)

References 38 publications

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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%

“…By counting the partitions with a positive fluorescent signal, the number of targets could be obtained without the need for a calibration curve. A variety of digital isothermal amplification techniques were developed for fast amplifying the target nucleic acids at a simple constant temperature and avoiding the complex process of thermal cycling of dPCR, such as digital loop-mediated isothermal amplification (LAMP) − or recombinase polymerase amplification (RPA). , By virtue of high sensitivity, high accuracy, satisfactory resistance to inhibitors, less amplification bias, and sample contamination, these “digital” techniques have flourished in the fields of nucleic acid detection at extremely low concentrations for analysis of pathogens, ,, cancer biomarkers, , rare mutations, methylation, gene expression, sequencing assistance, , etc. Hitherto, those digital assays are limited to fluorescence detection, mainly including fluorescence microscopy and flow cytometry, and few other techniques have been introduced to digital assay.…”

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confidence: 99%

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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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Section: Resultsmentioning

confidence: 99%

mentioning

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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“…Multiplexed biosensing assays give a clearer picture of valuable health information than single targets alone. Several examples of digital technology allow multiplexing of nucleic acids and proteins from a single sample; however, this research area is significantly smaller than that of studying new droplet-based technology [ 39 , 53 , 128 , 134 ].…”

Section: Discussionmentioning

confidence: 99%

“…Like in dELISA, the use of multiple fluorescent probes enables multiplexed detection in digital nucleic acid sensing. For example, a dLAMP assay was developed for detection of HCV and HIV by designing target specific fluorescent labeled primers that only activate when used in LAMP [ 134 ]. A real-time fluorescence curve recorded for the corresponding colors and quantified of multiple targets with high sensitivity of four copies/reaction.…”

Section: Digital Biosensing For Nucleic Acidsmentioning

confidence: 99%

Recent Advances in Digital Biosensing Technology

et al. 2022

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Digital biosensing assays demonstrate remarkable advantages over conventional biosensing systems because of their ability to achieve single-molecule detection and absolute quantification. Unlike traditional low-abundance biomarking screening, digital-based biosensing systems reduce sample volumes significantly to the fL-nL level, which vastly reduces overall reagent consumption, improves reaction time and throughput, and enables high sensitivity and single target detection. This review presents the current technology for compartmentalizing reactions and their applications in detecting proteins and nucleic acids. We also analyze existing challenges and future opportunities associated with digital biosensing and research opportunities for developing integrated digital biosensing systems.

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“…For complete details on the use and execution of this protocol, please refer to Tan et al. (2021a , 2021b) .…”

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confidence: 99%

Droplet microfluidic-based loop-mediated isothermal amplification (dLAMP) for simultaneous quantification of multiple targets

Tan

Wang

et al. 2022

STAR Protocols

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Multiplexed droplet loop-mediated isothermal amplification with scorpion-shaped probes and fluorescence microscopic counting for digital quantification of virus RNAs

Abstract: Highly sensitive digital nucleic acid techniques are of great significance for the prevention and control of epidemic diseases. Here we report the development of multiplex droplet loop-mediated isothermal amplification (multiplexed...

Cited by 28 publications

References 38 publications

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

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

Recent Advances in Digital Biosensing Technology

Droplet microfluidic-based loop-mediated isothermal amplification (dLAMP) for simultaneous quantification of multiple targets

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