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

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.

Download Full-text

DNA-templated copper nanoclusters obtained via TdT isothermal nucleic acid amplification for mercury(ii) assay

Analytical Methods ◽

10.1039/c9ay01214a ◽

2019 ◽

Vol 11 (32) ◽

pp. 4165-4172 ◽

Cited By ~ 2

Author(s):

Jing-Lin He ◽

Xing-Xing Wang ◽

Ting-Ting Mei ◽

Ling Wu ◽

Ju-Lan Zeng ◽

...

Keyword(s):

Nucleic Acid ◽

Fluorescence Sensor ◽

Isothermal Amplification ◽

Nucleic Acid Amplification ◽

Copper Nanoclusters ◽

Isothermal Nucleic Acid Amplification

The Hg2+ fluorescence sensor based on TdT isothermal amplification DNA templated CuNCs was successfully constructed.

Download Full-text

A novel exonuclease-assisted isothermal nucleic acid amplification with ultrahigh specificity mediated by full-length Bst DNA polymerase

Chemical Communications ◽

10.1039/c8cc04577a ◽

2018 ◽

Vol 54 (75) ◽

pp. 10562-10565 ◽

Cited By ~ 7

Author(s):

Xin Ye ◽

Yang Li ◽

Lijuan Wang ◽

Xueen Fang ◽

Jilie Kong

Keyword(s):

Nucleic Acid ◽

Nucleic Acids ◽

Dna Polymerase ◽

Isothermal Amplification ◽

Full Length ◽

Nucleic Acid Amplification ◽

Isothermal Nucleic Acid Amplification

A novel exonuclease-assisted isothermal amplification to amplify and determine nucleic acids very sensitively and with ultrahigh specificity.

Download Full-text

Strategies for Isothermal Amplification of Nucleic Acids: are they Ready to be Applied in Point of Care Diagnosis of Mycosis?

Biointerface Research in Applied Chemistry ◽

10.33263/briac113.1055910571 ◽

2020 ◽

Vol 11 (3) ◽

pp. 10559-10571

Keyword(s):

Nucleic Acid ◽

Clinical Laboratory ◽

Multiple Displacement Amplification ◽

Rolling Circle Amplification ◽

High Sensitivity ◽

Isothermal Amplification ◽

Nucleic Acid Amplification ◽

Clinical Samples ◽

Rolling Circle ◽

Isothermal Nucleic Acid Amplification

The early detection of invasive fungal infection (IFD) is significant in order to decrease mortality in susceptible patients. There is, therefore, a need for sensitive and specific fungal species detection assays in a clinical laboratory for early targeted therapy. The isothermal amplification method may be useful for the screening of fungal isolates, especially in resource-poor settings. Therefore, our aim was to review the isothermal nucleic acid amplification methods and their applications in fungal pathogen detection. Out of 50 reported studies, 28, 12, 6, 2, and 2 studies used the isothermal-based assays of a loop-mediated isothermal amplification (LAMP), nucleic acid sequence-based amplification (NASBA), rolling circle amplification (RCA), multiple displacement amplification (MDA) and polymerase Spiral Reaction (PSR), respectively. Thirty-two studies used clinical samples, 18 pure culture, and four environmental samples. The diagnostic accuracy of isothermal nucleic acid amplification testing for pathogenic fungal was reported as high (sensitivity 0.89–1.0 and specificity 0.63–1.0) in all studies irrespective of the sample tested. Although the isothermal-based assays showed high sensitivity and specificity in reported studies, it is still poorer than that of PCR assays. However, improving the assay to make it simpler, more effective, and inexpensive compared with newer PCR methods are still needed.

Download Full-text

Quantitative Isothermal Amplification on Paper Membranes using Amplification Nucleation Site Analysis

10.1101/2022.01.11.475898 ◽

2022 ◽

Author(s):

Benjamin P. Sullivan ◽

Yu-Shan Chou ◽

Andrew T. Bender ◽

Coleman D. Martin ◽

Zoe G. Kaputa ◽

...

Keyword(s):

Viral Load ◽

Nucleic Acid ◽

Infectious Diseases ◽

Point Of Care ◽

Nucleation Site ◽

Isothermal Amplification ◽

Nucleic Acid Amplification ◽

Hiv Viral Load ◽

Middle Income ◽

Nucleation Sites

Quantitative nucleic acid amplification tests (qNAATs) are critical in treating infectious diseases, such as in HIV viral load monitoring or SARS-CoV-2 testing, in which viral load indicates viral suppression or infectivity. Quantitative PCR is the gold standard tool for qNAATs; however, there is a need to develop point-of-care (POC) qNAATs to manage infectious diseases in outpatient clinics, low- and middle-income countries, and the home. Isothermal amplification methods are an emerging tool for POC NAATs as an alternative to traditional PCR-based workflows. Previous works have focused on relating isothermal amplification bulk fluorescence signals to input copies of target nucleic acids for sample quantification with limited success. In this work, we show that recombinase polymerase amplification (RPA) reactions on paper membranes exhibit discrete fluorescent amplification nucleation sites. We demonstrate that the number of nucleation sites can be used to quantify HIV-1 DNA and RNA in less than 20 minutes. An image-analysis algorithm quantifies nucleation sites and determines the input nucleic acid copies in the range of 67-3,000 copies per reaction. We demonstrate a mobile phone-based system for image capture and onboard processing, illustrating that this method may be used at the point-of-care for qNAATs with minimal instrumentation.

Download Full-text

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

10.1101/2021.08.18.456885 ◽

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.

Download Full-text

High-Surety Isothermal Amplification and Detection of SARS-CoV-2

mSphere ◽

10.1128/msphere.00911-20 ◽

2021 ◽

Vol 6 (3) ◽

Author(s):

Sanchita Bhadra ◽

Timothy E. Riedel ◽

Simren Lakhotia ◽

Nicholas D. Tran ◽

Andrew D. Ellington

Keyword(s):

Nucleic Acid ◽

False Negative ◽

Lateral Flow ◽

Isothermal Amplification ◽

Boolean Logic ◽

Nucleic Acid Amplification ◽

Rapid Diagnostics ◽

One Pot ◽

Loop Mediated Isothermal Amplification ◽

Isothermal Nucleic Acid Amplification

ABSTRACT Isothermal nucleic acid amplification tests (iNATs), such as loop-mediated isothermal amplification (LAMP), are good alternatives to PCR-based amplification assays, especially for point-of-care and low-resource use, in part because they can be carried out with relatively simple instrumentation. However, iNATs can often generate spurious amplicons, especially in the absence of target sequences, resulting in false-positive results. This is especially true if signals are based on non-sequence-specific probes, such as intercalating dyes or pH changes. In addition, pathogens often prove to be moving, evolving targets and can accumulate mutations that will lead to inefficient primer binding and thus false-negative results. Multiplex assays targeting different regions of the analyte and logical signal readout using sequence-specific probes can help to reduce both false negatives and false positives. Here, we describe rapid conversion of three previously described SARS-CoV-2 LAMP assays that relied on a non-sequence-specific readout into individual and multiplex one-pot assays that can be visually read using sequence-specific oligonucleotide strand exchange (OSD) probes. We describe both fluorescence-based and Boolean logic-gated colorimetric lateral flow readout methods and demonstrate detection of SARS-CoV-2 virions in crude human saliva. IMPORTANCE One of the key approaches to treatment and control of infectious diseases, such as COVID-19, is accurate and rapid diagnostics that is widely deployable in a timely and scalable manner. To achieve this, it is essential to go beyond the traditional gold standard of quantitative PCR (qPCR) that is often faced with difficulties in scaling due to the complexity of infrastructure and human resource requirements. Isothermal nucleic acid amplification methods, such as loop-mediated isothermal amplification (LAMP), have been long pursued as ideal, low-tech alternatives for rapid, portable testing. However, isothermal approaches often suffer from false signals due to employment of nonspecific readout methods. We describe general principles for rapidly converting nonspecifically read LAMP assays into assays that are read in a sequence-specific manner by using oligonucleotide strand displacement (OSD) probes. We also demonstrate that inclusion of OSD probes in LAMP assays maintains the simplicity of one-pot assays and a visual yes/no readout by using fluorescence or colorimetric lateral-flow dipsticks while providing accurate sequence-specific readout and the ability to logically query multiplex amplicons for redundancy or copresence. These principles not only yielded high-surety isothermal assays for SARS-CoV-2 but might also aid in the design of more sophisticated molecular assays for other analytes.

Download Full-text

Multicenter Clinical Evaluation of the Alere i Respiratory Syncytial Virus Isothermal Nucleic Acid Amplification Assay

Journal of Clinical Microbiology ◽

10.1128/jcm.01777-17 ◽

2017 ◽

Vol 56 (3) ◽

Cited By ~ 4

Author(s):

Ferdaus Hassan ◽

Lindsay M. Hays ◽

Aleta Bonner ◽

Bradley J. Bradford ◽

Ruffin Franklin ◽

...

Keyword(s):

Respiratory Syncytial Virus ◽

Nucleic Acid ◽

Care Setting ◽

Point Of Care ◽

Nucleic Acid Amplification ◽

Rt Pcr ◽

Pcr Assay ◽

Isothermal Nucleic Acid Amplification ◽

Syncytial Virus ◽

Respiratory Specimens

ABSTRACTThe Alere i respiratory syncytial virus (RSV) assay is an isothermal nucleic acid amplification test capable of detecting RSV directly from respiratory specimens, with results being available in ≤13 min after test initiation. The objective of this study was to evaluate the performance characteristics of the Alere i RSV assay in a point-of-care setting by using direct nasopharyngeal (NP) swab specimens (direct NP) and nasopharyngeal swab specimens eluted and transported in viral transport medium (VTM NP). The study was a prospective, multicenter, clinical trial conducted at 9 sites across the United States to evaluate the clinical performance of the Alere i RSV assay with respiratory specimens obtained from both children (age, <18 years) and older adults (age, >60 years). The performance of the Alere i RSV assay was compared with that of the reference method, the Prodesse ProFlu+ real-time reverse transcriptase PCR (RT-PCR) assay. All specimens with discrepant test results were tested further by a second FDA-cleared PCR assay (the Verigene respiratory virus plus nucleic acid test; Luminex Inc., TX). A total of 554 subjects with signs and symptoms of respiratory infections were enrolled, and respiratory samples were collected in this study. In comparison with the ProFlu+ real-time RT-PCR, the overall sensitivity and specificity of Alere i RSV assay for the detection of RSV were 98.6% (95% confidence interval [CI], 94.4 to 99.7%) and 98.0% (95% CI, 95.8 to 99.1%), respectively, for direct NP and 98.6% (95% CI, 94.4 to 99.7%) and 97.8% (95% CI, 95.5 to 98.9%), respectively, for VTM NP. The Alere i RSV is a highly sensitive and specific molecular assay ideal for rapid RSV detection in patients in the point-of-care setting due to its minimal hands-on time and rapid result availability.

Download Full-text

Isothermal nucleic acid amplification technologies for point-of-care diagnostics: a critical review

Lab on a Chip ◽

10.1039/c2lc40100b ◽

2012 ◽

Vol 12 (14) ◽

pp. 2469 ◽

Cited By ~ 399

Author(s):

Pascal Craw ◽

Wamadeva Balachandran

Keyword(s):

Nucleic Acid ◽

Critical Review ◽

Point Of Care ◽

Nucleic Acid Amplification ◽

Isothermal Nucleic Acid Amplification ◽

Point Of Care Diagnostics

Download Full-text

A novel method to control carryover contamination in isothermal nucleic acid amplification

Chemical Communications ◽

10.1039/c7cc06469a ◽

2017 ◽

Vol 53 (77) ◽

pp. 10696-10699 ◽

Cited By ~ 17

Author(s):

Cuiping Ma ◽

Fuxin Wang ◽

Xiudan Wang ◽

Lingzhi Han ◽

Hao Jing ◽

...

Keyword(s):

Nucleic Acid ◽

Restriction Endonuclease ◽

Isothermal Amplification ◽

Nucleic Acid Amplification ◽

Loop Mediated Isothermal Amplification ◽

Isothermal Nucleic Acid Amplification ◽

Recognition Sites ◽

Novel Method

We developed a novel method to control carryover contamination in loop-mediated isothermal amplification (LAMP) by primer engineering to carry recognition sites for a restriction endonuclease, providing a robust ability to eliminate carryover contaminants.

Download Full-text