Kinetics of Isothermal Dumbbell Exponential Amplification: Effects of Mix Composition on LAMP and Its Derivatives

Savonnet, Maud; Aubret, Mathilde; Laurent, Patricia; Roupioz, Yoann; Cubizolles, Myriam; Buhot, Arnaud

doi:10.3390/bios12050346

Cited by 10 publications

(4 citation statements)

References 56 publications

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“…A recent study developed a kinetic model for the exponential phase of LAMP, but did not detail the steps involved in initiating amplification ( 35 ). Similarly, others have proposed more complicated models based on investigating the distribution of different concatemer lengths and the effect on exponential amplification ( 11 ).…”

Section: Discussionmentioning

confidence: 99%

Kinetics of elementary steps in loop-mediated isothermal amplification (LAMP) show that strand invasion during initiation is rate-limiting

Dangerfield

Paik

Bhadra

et al. 2022

Nucleic Acids Research

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Loop-mediated isothermal amplification (LAMP) has proven to be easier to implement than PCR for point-of-care diagnostic tests. However, the underlying mechanism of LAMP is complicated and the kinetics of the major steps in LAMP have not been fully elucidated, which prevents rational improvements in assay development. Here we present our work to characterize the kinetics of the elementary steps in LAMP and show that: (i) strand invasion / initiation is the rate-limiting step in the LAMP reaction; (ii) the loop primer plays an important role in accelerating the rate of initiation and does not function solely during the exponential amplification phase and (iii) strand displacement synthesis by Bst-LF polymerase is relatively fast (125 nt/s) and processive on both linear and hairpin templates, although with some interruptions on high GC content templates. Building on these data, we were able to develop a kinetic model that relates the individual kinetic experiments to the bulk LAMP reaction. The assays developed here provide important insights into the mechanism of LAMP, and the overall model should be crucial in engineering more sensitive and faster LAMP reactions. The kinetic methods we employ should likely prove useful with other isothermal DNA amplification methods.

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Section: Discussionmentioning

confidence: 99%

Kinetics of elementary steps in loop-mediated isothermal amplification (LAMP) show that strand invasion during initiation is rate-limiting

Dangerfield

Paik

Bhadra

et al. 2022

Nucleic Acids Research

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“…It is a novel approach that uses complete genomes to create LAMP primers for a set of target genomes for rapid detection of foodborne pathogens, which yielded similar results to Primer explorer V5 with the same nucleotide sequences. Additionally, a theoretical approach was developed by Savonnet et al [ 30 ], who studied the dynamics of LAMP’s second stage from the fundamentals of reaction rates to deduce physio-chemical properties from the experimental signal development. The dumbbell structure used in the study skipped the first stage of the LAMP.…”

Section: Overview Of the Lamp Assaymentioning

confidence: 99%

LAMP-Based Point-of-Care Biosensors for Rapid Pathogen Detection

2022

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Seeking optimized infectious pathogen detection tools is of primary importance to lessen the spread of infections, allowing prompt medical attention for the infected. Among nucleic-acid-based sensing techniques, loop-mediated isothermal amplification is a promising method, as it provides rapid, sensitive, and specific detection of microbial and viral pathogens and has enormous potential to transform current point-of-care molecular diagnostics. In this review, the advances in LAMP-based point-of-care diagnostics assays developed during the past few years for rapid and sensitive detection of infectious pathogens are outlined. The numerous detection methods of LAMP-based biosensors are discussed in an end-point and real-time manner with ideal examples. We also summarize the trends in LAMP-on-a-chip modalities, such as classical microfluidic, paper-based, and digital LAMP, with their merits and limitations. Finally, we provide our opinion on the future improvement of on-chip LAMP methods. This review serves as an overview of recent breakthroughs in the LAMP approach and their potential for use in the diagnosis of existing and emerging diseases.

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“…Amplification is performed at a constant temperature, which decreases the length of the procedure because time is not spent heating and cooling. In addition, using some methods of isothermal amplification, the amount of DNA increases by more than double at each stage of amplification [3]; this reaction is faster than a PCR. The replacement of thermocycling, in some cases, also allows the use of simpler and portable equipment such as a thermostat instead of a thermocycler.…”

Section: Introductionmentioning

confidence: 99%

New Method of Isothermal, Hairpin Assisted, Primer Independent Amplification of DNA

Naberezhnov,

Alferov,

Kuzmin

et al. 2023

BioChem

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The isothermal amplification of nucleic acids refers to processes that quickly increase the amount of DNA at a constant temperature. These methods are mainly developed as alternatives to PCR for cases in which the application of a thermal cycler is not possible or the assay method must be as rapid as possible. We have developed a new method of isothermal amplification based on the formation of hairpins at the ends of DNA fragments containing palindromic sequences and increased by the hydrolysis of one or both DNA strands by restriction endonuclease, known as hairpin-assisted isothermal reaction (HAIR). The key steps in HAIR are the formation of a self-complementary hairpin and the DNA breakage introduced by nickase. The end hairpins facilitate primer-free amplification, the amplicon strand cleavage by nickase produces additional 3′ ends that serve as new amplification points, and the amount of DNA can increase exponentially. The rate of amplification in HAIR is more than five times the rate of loop-mediated isothermal amplification (LAMP), and the total amount of DNA product of HAIR is more than double the amount of the LAMP product.

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Kinetics of Isothermal Dumbbell Exponential Amplification: Effects of Mix Composition on LAMP and Its Derivatives

Cited by 10 publications

References 56 publications

Kinetics of elementary steps in loop-mediated isothermal amplification (LAMP) show that strand invasion during initiation is rate-limiting

Kinetics of elementary steps in loop-mediated isothermal amplification (LAMP) show that strand invasion during initiation is rate-limiting

LAMP-Based Point-of-Care Biosensors for Rapid Pathogen Detection

New Method of Isothermal, Hairpin Assisted, Primer Independent Amplification of DNA

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