scholarly journals A Robust, Low-Cost Instrument for Real-time Colorimetric Isothermal Nucleic Acid Amplification

2021 ◽  
Author(s):  
Frank Myers ◽  
Brian Moffatt ◽  
Ragheb El Khaja ◽  
Titash Chatterjee ◽  
Gurmeet Marwaha ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Point Of Care ◽  
Low Cost ◽  
Lamp Assay ◽  
Nucleic Acid Amplification ◽  
Diagnostic Assays ◽  
Isothermal Nucleic Acid Amplification ◽  
Point Of Care Diagnostics ◽  
Time Readout

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.

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

Sensors ◽  
2015 ◽  
Vol 15 (9) ◽  
pp. 23418-23430 ◽  
Author(s):  
Pascal Craw ◽  
Ruth Mackay ◽  
Angel Naveenathayalan ◽  
Chris Hudson ◽  
Manoharanehru Branavan ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Low Cost ◽  
Nucleic Acid Amplification ◽  
Isothermal Nucleic Acid Amplification

Long-term dry storage of enzyme-based reagents for isothermal nucleic acid amplification in a porous matrix for use in point-of-care diagnostic devices

The Analyst ◽  
2020 ◽  
Vol 145 (21) ◽  
pp. 6875-6886 ◽  
Author(s):  
Sujatha Kumar ◽  
Ryan Gallagher ◽  
Josh Bishop ◽  
Enos Kline ◽  
Joshua Buser ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Glass Fiber ◽  
Point Of Care ◽  
Porous Matrix ◽  
Isothermal Amplification ◽  
Nucleic Acid Amplification ◽  
Isothermal Nucleic Acid Amplification ◽  
Dry Storage

Long-term dry storage of enzyme-based isothermal amplification reagents in glass fiber porous matrix for use in point-of-care devices.

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

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

scholarly journals A comparative study of isothermal nucleic acid amplification methods for SARS-CoV-2 detection at point of care

2020 ◽  
Author(s):  
Diem Hong Tran ◽  
Hoang Quoc Cuong ◽  
Hau Thi Tran ◽  
Uyen Phuong Le ◽  
Hoang Dang Khoa Do ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Limit Of Detection ◽  
Direct Detection ◽  
Point Of Care ◽  
Nucleic Acid Amplification ◽  
Gold Standard Method ◽  
Synthetic Dna ◽  
Isothermal Nucleic Acid Amplification ◽  
Freeze Dried ◽  
Test Kit

ABSTRACTThe COVID-19, caused by the novel coronavirus SARS-CoV-2, has broken out of control all over the globe and put the majority of the world under lockdown. There have been no specific antiviral medications for SARS-CoV-2 while vaccines are still under development. Thus, rapid diagnosis and necessary public health measures are currently key parts to contain the pandemic. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is the gold standard method for SARS-CoV-2 detection. However, this method is not suitable for point-of-care (POC) diagnosis because of the timeconsuming procedure, the requirements of biosafety conditions and expensive equipment. In this study, the colorimetric isothermal nucleic acid amplification tests (iNAATs) for SARS-CoV-2 based on loop-mediated isothermal amplification (LAMP), cross-priming amplification (CPA), and polymerase spiral reaction (PSR) were developed and compared. The three methods exhibited similar performance with the limit of detection (LOD) as low as just 1 copy per reaction when evaluated on the synthetic DNA fragments. The results can be read with naked eyes within 30 minutes without crossreactivity to closely related coronaviruses. When tested with SARS-CoV-2 extracted genomic-RNA, LAMP outperformed both CPA and PSR assays. Moreover, the direct detection of SARS-CoV-2 in simulated patient samples (oropharyngeal and nasopharyngeal swabs) by colorimetric iNAATs was also successful. Further preparation of the lyophilized reagents for LAMP reactions revealed that the freeze-dried, ready-to-use kit maintained the sensitivity and LOD value of the liquid assays. These results strongly indicate that the colorimetric lyophilized LAMP test kit developed herein is highly suitable for detecting SARS-CoV-2 at POC.

scholarly journals Handyfuge-LAMP: low-cost and electricity-free centrifugation for isothermal SARS-CoV-2 detection in saliva

2020 ◽  
Author(s):  
Ethan Li ◽  
Adam Larson ◽  
Anesta Kothari ◽  
Manu Prakash
Keyword(s):  
Point Of Care ◽  
Low Cost ◽  
Lamp Assay ◽  
Sample Collection ◽  
Rna Detection ◽  
Open Hardware ◽  
Point Of Care Diagnostics ◽  
Simple Protocol ◽  
Specialized Equipment ◽  
The Cost

AbstractPoint of care diagnostics for COVID-19 detection are vital to assess infection quickly and at the source so appropriate measures can be taken. The loop-mediated isothermal amplification (LAMP) assay has proven to be a reliable and simple protocol that can detect small amounts of viral RNA in patient samples (<10 genomes per μL) (Nagamine, Hase, and Notomi 2002). Recently, Rabe and Cepko at Harvard published a sensitive and simple protocol for COVID-19 RNA detection in saliva using an optimized LAMP assay (Rabe and Cepko, 2020).This LAMP protocol has the benefits of being simple, requiring no specialized equipment; rapid, requiring less than an hour from sample collection to readout; and cheap, costing around $1 per reaction using commercial reagents. The pH based colorimetric readout also leaves little ambiguity and is intuitive. However, a shortfall in many nucleic acid-based methods for detection in saliva samples has been the variability in output due to the presence of inhibitory substances in saliva. Centrifugation to separate the reaction inhibitors from inactivated sample was shown to be an effective way to ensure reliable LAMP amplification. However, a centrifuge capable of safely achieving the necessary speeds of 2000 RPM for several minutes often costs hundreds of dollars and requires a power supply.We present here an open hardware solution- Handyfuge - that can be assembled with readily available components for the cost of <5 dollars a unit and could be used together with the LAMP assay for point of care detection of COVID-19 RNA from saliva. The device is then validated using the LAMP protocol from Rabe and Cepko. With the use of insulated coolers for reagent supply chain and delivery, the assay presented can be completed without the need for electricity or any laboratory scale infrastructure.

scholarly journals A simple check valve for microfluidic point of care diagnostics

Lab on a Chip ◽  
2016 ◽  
Vol 16 (22) ◽  
pp. 4436-4444 ◽  
Author(s):  
C. S. Ball ◽  
R. F. Renzi ◽  
A. Priye ◽  
R. J. Meagher
Keyword(s):  
Nucleic Acid ◽  
Point Of Care ◽  
Check Valve ◽  
Nucleic Acid Amplification ◽  
Nucleic Acid Amplification Testing ◽  
Point Of Care Diagnostics

Laser cut microfluidic check valves enable staged reagent delivery, pumping, and point of care nucleic acid amplification testing.

scholarly journals A robust, hand-powered, instrument-free sample preparation system for point-of-care pathogen detection

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Fei Zhao ◽  
Eun Yeong Lee ◽  
Geun Su Noh ◽  
Jaehyup Shin ◽  
Huifang Liu ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Sample Preparation ◽  
Pathogen Detection ◽  
Point Of Care ◽  
Low Cost ◽  
Sample Pretreatment ◽  
Sample Volume ◽  
Nucleic Acid Isolation ◽  
Point Of Care Diagnostics ◽  
Reaction Matrix

Abstract Here, we describe a simple, universal protocol for use in nucleic acid testing-based pathogen diagnostics, which requires only hand-powered sample preparation, including the processes of pathogen enrichment and nucleic acid isolation. The protocol uses low-cost amine-functionalized diatomaceous earth with a 1-μm Teflon filter as a reaction matrix in both stages of the process, using homobifunctional imidoesters. Using a simple syringe as a pump, the capture efficiency for a large sample volume (<50 mL) was enhanced by up to 98.3%, and the detection limit was 1 CFU/mL, 100-fold better than that of common commercial nucleic acid isolation kit. This protocol can also be combined with commercialized 96-well filter plates for robust sample preparation. Our proposed system is robust, simple, low-cost, universal, and rapid (taking <20 min), and it works regardless of the ambient environment and sample pretreatment, requiring no electricity or instruments. Its benefits include the simplicity of producing its components and its ease of operation, and it can be readily integrated with other assays for point-of-care diagnostics.

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

2017 ◽  
Vol 9 (18) ◽  
pp. 15245-15253 ◽  
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

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

2006 ◽  
Vol 52 (4) ◽  
pp. 624-633 ◽  
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 &gt;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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