Mathematical model to reduce loop mediated isothermal amplification (LAMP) false‐positive diagnosis

Abstract

Loop mediated isothermal amplification (LAMP) is a nucleic acid amplification technique performed under isothermal conditions. The output of this amplification technique includes multiple different sizes of deoxyribonucleic acid (DNA) structures which are identified by a banding pattern on gel electrophoresis plots. Although this is a specific amplification technique, the complexity of the primer design and amplification still lead to the issue of obtaining false‐positive results, especially when a positive reading is determined solely by whether there is any banding pattern in the gel electrophoresis plot. Here, we first performed extensive LAMP experiments and evaluated the DNA structures using microchip electrophoresis. We then developed a mathematical model derived from the various components that make up an entire LAMP structure to predict the full LAMP structure size in base pairs. This model can be implemented by users to make predictions for specific, DNA size dependent, banding patterns on their gel electrophoresis plots. Each prediction is specific to the target sequence and primers used and therefore reduces incorrect diagnosis errors through identifying true‐positive and false‐positive results. This model was accurately tested with multiple primer sets in house and was also translatable to different DNA and RNA types in previously published literature. The mathematical model can ultimately be used to reduce false‐positive LAMP diagnosis errors for applications ranging from tuberculosis diagnostics to E. coli to numerous other infectious diseases.

Real-time kinetics and high-resolution melt curves in single-molecule digital LAMP to differentiate and study specific and non-specific amplification

“…in a tube) in the absence of template can be informative to provide information on performance of non-specific amplification. Another method to identify non-specific amplification includes mathematical modeling in conjunction with electrophoresis to distinguish between non-specific and specific banding patterns (9). However, in the presence of template, although specific and non-specific reactions occur simultaneously, they cannot be monitored simultaneously.…”

Crispr-cas 12a combination to alleviate the false-positive in loop-mediated isothermal amplification-based diagnosis of Neisseria meningitidis

“…Single-use of LAMP embeds high risk of false-positive signals that challenge the employing LAMP-based assays into clinical practices [7,9]. However, LAMP has strong intrinsic ampli cation potential and simple to operate, hence would bene t the communities.…”

“…LAMP can gain PCR's sensitivity without complicated thermocycling, some LAMP assays can complete within 10 minutes. However, LAMP detection step acquired non-speci c indicators (such as Mg2+, intercalating dyes, labelled primers) that cannot distinguish spurious amplicons [7][8][9]. We documented several phenomena that real-time PCR protocols [4,10] could not recapitulate positive results gained by LAMP reactions and some LAMP positive cases lacked meningitidis speci c clinical symptoms [1].…”

“…We suspected that LAMP assay might acquire given false positive potentials [7][8][9]. With abilities to accurately recognize speci c sequence, the CRISPR/Cas system holds promising potentials.…”

A stoichiometric and pseudo kinetic model of loop mediated isothermal amplification

“…Extremely limited literature existing on theoretical knowledge about LAMP includes development of a method for better quantification of rise times for LAMP reactions [17] , demonstration of the effect of internal primer-template mismatches on LAMP efficiency [20] , and observation of the impact of primer dimers and self-amplifying hairpins on LAMP reactions and proposing a thermodynamic parameter to predict non-specific amplification for different sets of LAMP primers [21] . A recent study presented a mathematical model to reduce false-positive diagnosis by LAMP by predicting the expected size of amplicons and comparing it with the results obtained from electropherograms [22] . The authors presented a new method to analyse the increasing length of LAMP amplicons.…”