Visual Detection of Multiple Nucleic Acids in a Capillary Array

Jianwei Chen; Ning Shao; Jiaying Hu; Rong Li; Yuanshou Zhu; Dabing Zhang; Shujuan Guo; Junhou Hui; Peng Liu; Litao Yang; Sheng-Ce Tao

doi:10.3791/56597

Method Article

Visual Detection of Multiple Nucleic Acids in a Capillary Array

DOI:

10.3791/56597

⸱

November 15th, 2017

Jianwei Chen*¹^,²^,³ , Ning Shao*¹^,²^,³ , Jiaying Hu*⁴ , Rong Li⁴ , Yuanshou Zhu¹^,²^,³ , Dabing Zhang⁴^,⁵ , Shujuan Guo¹^,²^,³ , Junhou Hui⁶ , Peng Liu⁶ , Litao Yang⁴ , Sheng-Ce Tao¹^,²^,³

¹Key Laboratory of Systems Biomedicine (Ministry of Education), Shanghai Center for Systems Biomedicine, Shanghai Jiao Tong University, ²State Key Laboratory of Oncogenes and Related Genes, ³School of Biomedical Engineering, Shanghai Jiao Tong University, ⁴Collaborative Innovation Center for Biosafety of GMOs, National Center for the Molecular Characterization of Genetically Modified Organisms, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, ⁵Key Laboratory of Crop Marker-Assisted Breeding of Huaian Municipality, Jiangsu Collaborative Innovation Center of Regional Modern Agriculture and Environmental Protection, ⁶Department of Biomedical Engineering, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, School of Medicine, Tsinghua University

Summary

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This protocol describes the fabrication of a small, ready-to-use cassette that can be applied for visual detection of multiple nucleic acids in a single, test that is easy to operate. In this approach, a capillary array was used for multiplex and highly efficient detection of GMO targets.

Abstract

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Multi-target, short time, and resource-affordable methodologies for the detection of multiple nucleic acids in a single, easy to operate test are urgently needed in disease diagnosis, microbial monitoring, genetically modified organism (GMO) detection, and forensic analysis. We have previously described the platform called CALM (Capillary Array-based Loop-mediated isothermal amplification for Multiplex visual detection of nucleic acids). Herein, we describe improved fabrication and performance processes for this platform. Here, we apply a small, ready-to-use cassette assembled by capillary array for multiplex visual detection of nucleic acids. The capillary array is pre-treated into a hydrophobic and hydrophilic pattern before fixing loop-mediated isothermal amplification (LAMP) primer sets in capillaries. After assembly of the loading adaptor, LAMP reaction mixture is loaded and isolated into each capillary, due to capillary force by a single pipetting step. The LAMP reactions are performed in parallel in the capillaries. The results are visually read out by illumination with a hand-held UV flashlight. Using this platform, we demonstrate monitoring of 8 frequently appearing elements and genes in GMO samples with high specificity and sensitivity. In summary, the platform described herein is intended to facilitate the detection of multiple nucleic acids. We believe it will be widely applicable in fields where high-throughput nucleic acid analysis is required.

Introduction

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Low-cost, quick, and easy to use systems for the simultaneous detection of multiple nucleic acids are urgently needed in a wide range of fields, such as clinical diagnostics¹^,²^,³, GMO detection⁴^,⁵^,⁶, microbial monitoring⁷^,⁸^,⁹, forensic analysis¹⁰^,¹¹, and especially point-of-care tests (POCTs), where resources are usually limited

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Protocol

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NOTE: This protocol assumes that the stainless steel mold bearing the shape for the desired micro-channels and the loading adaptor have already been made (3D files are provided as Supplemental Files 1 and 2). This protocol also assumes that plant DNA isolation has already been carried out.

1. Fabrication of the Capillary Array-based Ready-to-use Cassette

Clean the stainless-steel mold.
1. Wash the stainless-steel mold by detergent, ethanol, and deionized water to remove the potential contaminants. Dry the mold by nitrogen.
Clean the capil....

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Results

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In this method, it is important to prevent cross-contamination among different capillaries during the sample loading. For this purpose, chitosan was introduced, which could retain the primers in individual capillaries. To test whether it worked or not, we pre-fixed the ADH1 (endogenous reference gene of maize) primer set in the capillary cassette with the pattern of "T" and "U", as illustrated in Figure 3a. As expected, only .......

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Discussion

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The CALM platform demonstrated here, which combines the LAMP technology with a capillary array, enables the simultaneous detection of multiple GMO-related gene targets in a single, highly effective and easy to operate test.

To successfully perform the multiplex LAMP reactions in the cassette, three critical points need to be noticed. Firstly, achieving the same height for the upper side of the capillaries and the hydrophilic and hydrophobic pattern of the capillary array are critical for simul.......

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Disclosures

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The authors have nothing to disclose.

Acknowledgements

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This study was funded in part by National Natural Science Foundation of China Grants (31370813, 3147670, 31670831 and 31600672,), the National Transgenic Plant Special Fund (2016ZX08012-003, 2016ZX08012-005), Program for New Century Excellent Talents in University, the National Key Research and Development Project of China (2016YFA0500601) and China Postdoctoral Science Foundation (2016M591667).

....

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Materials

List of materials used in this article
Name	Company	Catalog Number	Comments
UltraEverDry(super-hydrophobic coat)	UltraTech	4001	supplier:Exiron chemistry(CHINA) CO.,LTD.
PDMS	Dow Corning	8332557
Bst polymerase	New England BioLabs	M0275L
betain	Sigma-Aldrich	B0300-1VL
calcein	Sigma-Aldrich	C0875-5G
MnCl₂	Sigma-Aldrich	MKBP0495V
MgSO₄	New England BioLabs	B1003S
dNTPs	Shanghai Sangon	B804BA0022
chitosan	Shanghai Sangon	LJ0805S309J
Photoshop 7.0 software	Adobe Systems Inc., CA, USA		Image analysis
GenePix Pro 6.1	Molecular Devices, CA, USA		microarray analysis software
AutoCAD	Adobe Systems Inc.		3D construction software
UV filter (ZWB2)	YXSensing		supplier : taobao

References

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Urdea, M., et al. Requirements for high impact diagnostics in the developing world. Nature. 444, Suppl 1. 73-79 (2006).
Yager, P., Domingo, G. J., Gerdes, J. Point-of-care diagnostics for global health. Annu Rev Biomed Eng. 10, 107-144 (2008).
Opota, O., Jaton,....

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Visual Detection of Multiple Nucleic Acids in a Capillary Array

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Summary

Abstract

Introduction

Protocol

Results

Discussion

Disclosures

Acknowledgements

Materials

References

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