Submitted to: Molecules
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/1/2015
Publication Date: 4/7/2015
Publication URL: http://handle.nal.usda.gov/10113/60735
Citation: Wang, D., Brewster, J.D., Paul, M., Tomasula, P.M. 2015. Two methods for increased specificity and sensitivity in loop-mediated isothermal amplification. Molecules. 20:4048-6059. DOI: 10.3390/molecules20046048.
Interpretive Summary: Consumption of raw milk is slowly growing in the US due to assumptions that it is more nutritious and easier to digest than pasteurized milk. Pasteurization is a heat-treatment applied to milk which eliminates pathogens and assures its safety. Listeria monocytogenes, one of the pathogens that has been found in raw milk, causes listeriosis, which poses the highest risk of serious disease or death to the very young, the elderly, pregnant women and those with weakened immune systems. In this study, we developed an improvement to an assay known as LAMP, a useful tool for rapid detection of Listeria monocytogenes. The improved assay is more sensitive and specific than previous versions. This information will help advance efforts to develop rapid methods for detection of pathogens to prevent human illness caused by foodborne disease.
Technical Abstract: The technique of loop-mediated isothermal amplification (LAMP) utilizes 4 (or 6) primers targeting 6 (or 8) 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 assays by Tang, et al.