Two Methods for Increased Specificity and Sensitivity in Loop-Mediated Isothermal Amplification

Abstract

The technique of loop-mediated isothermal amplification (LAMP) utilizes four (or six) primers targeting six (or eight) regions within a fairly small segment of a genome for amplification, with concentration higher than that used in traditional PCR methods. The high concentrations of primers used leads to an increased likelihood of non-specific amplification induced by primer dimers. In this study, a set of LAMP primers were designed targeting the prfA gene sequence of Listeria monocytogenes, and dimethyl sulfoxide (DMSO) as well as Touchdown LAMP were employed to increase the sensitivity and specificity of the LAMP reactions. The results indicate that the detection limit of this novel LAMP assay with the newly designed primers and additives was 10 fg per reaction, which is ten-fold more sensitive than a commercial Isothermal Amplification Kit and hundred-fold more sensitive than previously reported LAMP assays. This highly sensitive LAMP assay has been shown to detect 11 strains of Listeria monocytogenes, and does not detect other Listeria species (including Listeria innocua and Listeria invanovii), providing some advantages in specificity over commercial Isothermal Amplification Kits and previously reported LAMP assay.

A loop-mediated isothermal amplification method for rapid direct detection and differentiation of nonpathogenic and verocytotoxigenicEscherichia coliin beef and bovine faeces

“…(), or perhaps a touchdown LAMP approach could be investigated, as was reported recently by Wang et al . (). However, it should be noted that after an enrichment step quantification of E. coli or VTEC would not be possible any more.…”

Loop-mediated isothermal amplification (LAMP) coupled with bioluminescence for the detection of Listeria monocytogenes at low levels on food contact surfaces

“…Similarly, Martinon et al (2012) reported that the real-time molecular methods (PCR, PMA-PCR) overestimated L. monocytogenes counts on environmental surfaces compared to the culture technique. However, the occurrence of false-positive results may be significantly reduced by avoiding cross-contamination, high humidity and temperatures, when handling reaction tubes and inadequate reaction volumes (Bird et al, 2013;Wang et al, 2015). Nevertheless, apart from a few disadvantages of LAMP technique, it was equally effective as reference ISO method for L. monocytogenes detection, but it reduced significantly the total time of analysis (from minimum 5 days for ISO to less than 2 days including the enrichment time).…”

“…Usually, in order to amplify six to eight target DNA regions, the LAMP employs from four to six primers with much higher concentration than that used in traditional PCR-based methods, which may lead to an increased probability of non-specific amplification induced by formed primer dimers, giving the false positive results (Wang, Brewster, Paul, & Tomasula, 2015). Another explanation might be due to the possibility of DNA amplification of lethally injured or dead cells, which can be undetectable by ISO method (Lim, Zheng, Mik s-Krajnik, Turner, & Yuk, 2015).…”

Rapid sex determination of a wild passerine species using loop‐mediated isothermal amplification (LAMP)

“…Consequently, one of our goals was to find one single incubation temperature that would work accurately for both CHD-W and CHD-Z primer sets so that separate reactions could be run simultaneously on the same thermoblock, requiring less equipment and time overall. False-positive results may also be obtained through primer-dimer formation, which is more likely to occur when high concentrations of primers are used, as are in a LAMP assay (Meagher, Priye, Light, Huang, & Wang, 2018 (Ball et al, 2016;Hsieh et al, 2014;Wang, Brewster, Paul, & Tomasula, 2015). Therefore, The most commonly reported drawback of LAMP is high rates of false positives.…”