Visual Detection of Isothermal Nucleic Acid Amplification Using pH-Sensitive Dyes

Tanner, Nathan A.; Zhang, Yinhua; Evans, Thomas C.

doi:10.2144/000114253

Cited by 526 publications

(479 citation statements)

References 26 publications

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“…44 LAMP was performed using the Bst 2.0 DNA Polymerase, an in silico designed homologue of Bst DNA Polymerase I, Large Fragment, commonly used in this reaction, and its supplied buffer. 45 In transitioning the reaction from tubes to paper, the concentration of betaine, primers, and enzyme were optimized, as was the incubation time, to minimize nonspecific reactions and maximize sensitivity, as shown in Figure S1 and discussed in the Supporting Information. The final reactions contained 1× isothermal amplification buffer (10 mM Tris-HCl, 10 mM (NH 4 ) 2 SO 4 , 50 mM KCl, 2 mM MgSO 4 , 0.1% Tween-20) supplemented with an additional 6 mM MgSO 4 , 0.9 M betaine, 1.4 mM of each dNTP, 1.6 μM of each inner primer (FIP, BIP), 0.8 μM of each loop primer (LF, LB), 0.2 μM of each outer primer (F3, B3), and 0.32 U of polymerase.…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

“Paper Machine” for Molecular Diagnostics

2015

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Clinical tests based on primer-initiated amplification of specific nucleic acid sequences achieve high levels of sensitivity and specificity. Despite these desirable characteristics, these tests have not reached their full potential because their complexity and expense limit their usefulness to centralized laboratories. This paper describes a device that integrates sample preparation and loop-mediated isothermal amplification (LAMP) with end point detection using a hand-held UV source and camera phone. The prototype device integrates paper microfluidics (to enable fluid handling) and a multilayer structure, or a "paper machine", that allows a central patterned paper strip to slide in and out of fluidic path and thus allows introduction of sample, wash buffers, amplification master mix, and detection reagents with minimal pipetting, in a hand-held, disposable device intended for point-of-care use in resource-limited environments. This device creates a dynamic seal that prevents evaporation during incubation at 65°C for 1 h. This interval is sufficient to allow a LAMP reaction for the Escherichia coli malB gene to proceed with an analytical sensitivity of 1 doublestranded DNA target copy. Starting with human plasma spiked with whole, live E. coli cells, this paper demonstrates full integration of sample preparation with LAMP amplification and end point detection with a limit of detection of 5 cells. Further, it shows that the method used to prepare sample enables concentration of DNA from sample volumes commonly available from fingerstick blood draw.

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Section: ■ Materials and Methodsmentioning

confidence: 99%

“Paper Machine” for Molecular Diagnostics

2015

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“…The gel electrophoretic detection of LAMP product is more time consuming, but this phase can be spared when dsDNA binding SybrGreen or pH sensitive dyes are used in the reaction. The naked eye can distinguish between positive and negative LAMP reactions based on colour alteration (TANNER et al, 2015).…”

Section: Resultsmentioning

confidence: 99%

Loop-mediated isothermal amplification based approach as an alternative to recombinase polymerase amplification based detection of Mangalitza component in food products

Zsolnai

Szántó-Egész²,

Ferencz-Elblinger

et al. 2017

Acta Alimentaria

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We used an alternative approach, loop-mediated isothermal amplifi cation, to detect Mangalitza component in food products, and it has been compared to an established Recombinase Polymerase Amplifi cation test. The correlation between the assays was signifi cant (P<0.01). Linear determination coeffi cient between the assays was 0.993 and level of diagnostic agreement was high (Kappa=0.971).Previously, a real-time PCR method based on TaqMan probe was developed for detection of Mangalitza meat in food products, using a Mangalitza specifi c sequence. Other Mangalitza specifi c sequences suitable for the same purpose are also in use (V. STÉGER, personal communication).Approaches like real-time monitoring of accumulation of the specifi c DNA product usually require specialised laboratory equipment. For Mangalitza detection, portable Recombinase Polymerase Amplifi cation (RPA) approach has been developed (SZÁNTÓ-EGÉSZ et al., 2016), which requires a device capable of maintaining 39 °C and a lateral fl ow strip with easy yes/no indication of the successful amplifi cation.We wanted to develop another fast, non-PCR based test with minimal laboratory requirement to provide a third possibility to detect Mangalitza component in food.Keywords: food, LAMP, RPA, MangalitzaNowadays, a wide range of non-PCR amplifi cations are available, such as Helicase Dependent Amplifi cation (VINCENT et al., 2004), Transcription Mediated Amplifi cation (GUATELLI et al., 1990), Self-Sustained Sequence Replication (3SR) (GUATELLI et al., 1990), Rolling Circle Amplifi cation (FIRE and XU, 1995), Standard Displacement Amplifi cation (SDA) (WALKER et al., 1992), and loop-mediated isothermal amplifi cation (LAMP) (NOTOMI et al., 2000). They usually do not require expensive instrumentation and have comparable diagnostic power to * To whom correspondence should be addressed. E-mail: attila.zsolnai@gmail.com This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License (https://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted use, distribution, and reproduction in any medium for non-commercial purposes, provided the original author and source are credited, a link to the CC License is provided, and changes -if any -are indicated.

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“…Given the high concentration of Mn 2ϩ that we found necessary to reduce background calcein fluorescence, it is unsurprising that the LAMP assay does not achieve the same sensitivity as the benchmark real-time PCR method. Further optimization to reduce the Mn 2ϩ concentration or implement an alternative detection method such as the use of hydroxynaphthol blue or pH-sensitive dyes could improve results to achieve comparable sensitivity (17,31).…”

Section: Discussionmentioning

confidence: 99%

Isothermal Detection of Mycoplasma pneumoniae Directly from Respiratory Clinical Specimens

et al. 2015

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Mycoplasma pneumoniae is a leading cause of community-acquired pneumonia (CAP) across patient populations of all ages. We have developed a loop-mediated isothermal amplification (LAMP) assay that enables rapid, low-cost detection of M. pneumoniae from nucleic acid extracts and directly from various respiratory specimen types. The assay implements calcein to facilitate simple visual readout of positive results in approximately 1 h, making it ideal for use in primary care facilities and resourcepoor settings. The analytical sensitivity of the assay was determined to be 100 fg by testing serial dilutions of target DNA ranging from 1 ng to 1 fg per reaction, and no cross-reactivity was observed against 17 other Mycoplasma species, 27 common respiratory agents, or human DNA. We demonstrated the utility of this assay by testing nucleic acid extracts (n ‫؍‬ 252) and unextracted respiratory specimens (n ‫؍‬ 72) collected during M. pneumoniae outbreaks and sporadic cases occurring in the United States from February 2010 to January 2014. The sensitivity of the LAMP assay was 88.5% tested on extracted nucleic acid and 82.1% evaluated on unextracted clinical specimens compared to a validated real-time PCR test. Further optimization and improvements to this method may lead to the availability of a rapid, cost-efficient laboratory test for M. pneumoniae detection that is more widely available to primary care facilities, ultimately facilitating prompt detection and appropriate responses to potential M. pneumoniae outbreaks and clusters within the community. Mycoplasma pneumoniae is a human pathogen that accounts for an estimated 15% to 20% of cases of community-acquired pneumonia (CAP) (1, 2). An atypical bacterial pneumonia, M. pneumoniae infection can result in both pulmonary and extrapulmonary manifestations, including pharyngitis, encephalitis, Stevens-Johnson syndrome, septic arthritis, and pericarditis, among others (3). Ongoing endemic M. pneumoniae infection is overlaid with cyclical epidemics of disease, typically occurring every 3 to 5 years (1). Although symptoms of M. pneumoniae infection are generally mild or subclinical, the organism accounts for up to 33% of hospitalizations among children and adults with bacterial pneumonia (4). In the absence of a diagnostic result, empirical therapy can be misdirected, resulting in inappropriate use of antibiotics and continued spread of M. pneumoniae in the community. This may promote the evolution of antibiotic resistance and leads to negative economic impact due to health care costs and missed school and work days.Traditional diagnostics for M. pneumoniae infections exist but are underused due to their cost, time-intensive nature, or requirement for specialized expertise and equipment. M. pneumoniae is fastidious and slow-growing, with isolation from liquid culture requiring up to 8 weeks and often resulting in low yield (5, 6). Appropriate interpretation of serological data requires paired acute-phase and convalescent-phase serum samples, constraining their use to retrosp...

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Visual Detection of Isothermal Nucleic Acid Amplification Using pH-Sensitive Dyes

Cited by 526 publications

References 26 publications

“Paper Machine” for Molecular Diagnostics

“Paper Machine” for Molecular Diagnostics

Loop-mediated isothermal amplification based approach as an alternative to recombinase polymerase amplification based detection of Mangalitza component in food products

Isothermal Detection of Mycoplasma pneumoniae Directly from Respiratory Clinical Specimens

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