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. 2022 Apr 18;3(2):101335.
doi: 10.1016/j.xpro.2022.101335. eCollection 2022 Jun 17.

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

Ya-Ling Tan  1   2 , Tao Wang  2 , Jianjun He  2 , Jian-Hui Jiang  2
Affiliations

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

Ya-Ling Tan et al. STAR Protoc. .

Abstract

The quantification of trace nucleic acids in biological samples is a frequent requirement in experimental and clinical diagnostics. Here, we present a protocol for the digital quantification of multiple nucleic acid targets with droplet microfluidics-based loop-mediated isothermal amplification (dLAMP). Our protocol provides a fundamental platform for the absolute quantification of multiple nucleic acid targets with high specificity, allowing readily adaption in various in vitro diagnostic settings. For complete details on the use and execution of this protocol, please refer to Tan et al. (2021a, 2021b).

Keywords: Health Sciences; Microscopy; Molecular/Chemical Probes.

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Conflict of interest statement

The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
Illustration of SP-based LAMP reaction Eight distinct regions are designated on the target DNA, labeled F3, F2, LFc, F1, B1c, LB, B2c, and B3 from the 5′ end (‘c’ represents a complementary region, hollow regions are complemented to the fulfilled regions of the same colors). Inner primers FIP/BIP consist of the F1c/B1c sequence and the F2/B2 sequence. F3/B3, LF, SP stand for outer primers, loop forward primer, and scorpion-shaped probe, respectively. FIP/BIP: Initiating the replicative DNA synthesis by the Bst DNA polymerase, extending and assisting the template destruction, playing a major role in the cycling amplification step. F3/B3: Displacing the newly synthesized DNA strand by FIP/BIP and releasing the target DNA. LF: Hybridizing the stem-loops and accelerating the LAMP reaction. SP: Generating fluorescence signal and accelerating the LAMP reaction.
Figure 2
Figure 2
Mask design of microfluidic chips (A) The Y-shape droplet microfluidic chip (Notes: The two oil inlets can be merged into one). (B) The droplet counting microwell chip.
Figure 3
Figure 3
Image of droplet generation (top) and counting microwell (bottom) chips
Figure 4
Figure 4
Gel images of LAMP reaction products Lane M: Marker; lane 1: blank; lane 2:HeLa cell genomic DNA; lane 3: HCV template; lane 4: cDNA from HCV-infected sample; lane 5: HIV template; lane 6: cDNA from HIV-infected sample.
Figure 5
Figure 5
Fluorescence images and intensity profiles of droplets in different channels Scale bar: 200 μm.
Figure 6
Figure 6
Whole-area images of droplet counting microwell chip Top: FITC channel, bottom: bright-field channel.
Figure 7
Figure 7
Droplet counting (A) Fluorescence images of droplets. Scale bar: 200 μm. (B) Automated measurement using NIS-Elements software. (C) Selected and segmented droplets after automated measurement. Scale bar: 200 μm. (D) Analysis of droplet count results.
Figure 8
Figure 8
Scatter plot of fluorescence intensities of droplets (A) FITC channel. (B) TAMRA channel. (C) Cy5 channel.
See this image and copyright information in PMC

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