A Simple, Low-Cost Platform for Real-Time Isothermal Nucleic Acid Amplification

The COVID-19 pandemic has highlighted the need for broader access to molecular diagnostics. Colorimetric isothermal nucleic acid amplification assays enable simplified instrumentation over more conventional PCR diagnostic assays and, as such, represent a promising approach for addressing this need. In particular, colorimetric LAMP (loop-mediated isothermal amplification) has received a great deal of interest recently. However, there do not currently exist robust instruments for performing these kinds of assays in high throughput with real-time readout of amplification signals. To address this need, we developed LARI, the LAMP Assay Reader Instrument. We have deployed over 50 LARIs for routine use in R&D and production environments, with over 12,000 assays run to date. In this paper, we present the design and construction of LARI along with thermal, optical, and assay performance characteristics. LARI can be produced for under $1500 and has broad applications in R&D, point-of-care diagnostics, and global health.

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Low-Cost, Open-Source Device for High-Performance Fluorescence Detection of Isothermal Nucleic Acid Amplification Reactions

ACS Biomaterials Science & Engineering ◽

10.1021/acsbiomaterials.1c01105 ◽

2021 ◽

Author(s):

Andrew H. Buultjens ◽

Koen Vandelannoote ◽

Liam K. Sharkey ◽

Benjamin P. Howden ◽

Ian R. Monk ◽

...

Keyword(s):

Nucleic Acid ◽

Open Source ◽

Fluorescence Detection ◽

High Performance ◽

Low Cost ◽

Nucleic Acid Amplification ◽

Isothermal Nucleic Acid Amplification

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Graphene Nanoprobes for Real-Time Monitoring of Isothermal Nucleic Acid Amplification

ACS Applied Materials & Interfaces ◽

10.1021/acsami.7b01134 ◽

2017 ◽

Vol 9 (18) ◽

pp. 15245-15253 ◽

Cited By ~ 17

Author(s):

Fan Li ◽

Xiaoguo Liu ◽

Bin Zhao ◽

Juan Yan ◽

Qian Li ◽

...

Keyword(s):

Nucleic Acid ◽

Real Time ◽

Nucleic Acid Amplification ◽

Real Time Monitoring ◽

Isothermal Nucleic Acid Amplification

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Antiprimer Quenching-Based Real-Time PCR and Its Application to the Analysis of Clinical Cancer Samples

Clinical Chemistry ◽

10.1373/clinchem.2005.063321 ◽

2006 ◽

Vol 52 (4) ◽

pp. 624-633 ◽

Cited By ~ 26

Author(s):

Jin Li ◽

Fengfei Wang ◽

Harvey Mamon ◽

Matthew H Kulke ◽

Lyndsay Harris ◽

...

Keyword(s):

Nucleic Acid ◽

Genetic Analysis ◽

Real Time ◽

Real Time Pcr ◽

Low Cost ◽

Pcr Primers ◽

Medical Diagnostics ◽

Nucleic Acid Amplification ◽

Clinical Samples ◽

Correct Identification

Abstract Background: Nucleic acid amplification plays an increasingly important role in genetic analysis of clinical samples, medical diagnostics, and drug discovery. We present a novel quantitative PCR technology that combines the advantages of existing methods and allows versatile and flexible nucleic acid target quantification in clinical samples of widely different origin and quality. Methods: We modified one of the 2 PCR primers by use of an oligonucleotide “tail” fluorescently labeled at the 5′ end. An oligonucleotide complementary to this tail, carrying a 3′ quenching molecule (antiprimer), was included in the reaction along with 2 primers. After primer extension, the reaction temperature was lowered such that the antiprimer hybridizes and quenches the fluorescence of the free primer but not the fluorescence of the double-stranded PCR product. The latter provides real-time fluorescent product quantification. This antiprimer-based quantitative real-time PCR method (aQRT-PCR) was used to amplify and quantify minute amounts of input DNA for genes important to cancer. Results: Simplex and multiplex aQRT-PCR demonstrated linear correlation (r2 >0.995) down to a DNA input equivalent to 20 cells. Multiplex aQRT-PCR reliably identified the HER-2 gene in microdissected breast cancer samples; in formalin-fixed, paraffin-embedded specimens; and in plasma circulating DNA from cancer patients. Adaptation to multiplex single-nucleotide polymorphism detection via allele-specific aQRT-PCR allowed correct identification of apolipoprotein B polymorphisms in 51 of 51 human specimens. Conclusion: The simplicity, versatility, reliability, and low cost of aQRT-PCR make it suitable for genetic analysis of clinical specimens.

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