Novel primers for increased specificity and sensitivity for the detection of Staphylococcus aureus by real-time LAMP

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 potential for greater specificity than two primer methods such as polymerase chain reaction. In this study, a set of LAMP primers was designed targeting the 16S-23S ribosomal RNA intergenic spacer region of Staphylococcus aureus, and tested for sensitivity and specificity in real-time LAMP reactions. Under optimized conditions, the detection limit of our developed assay was 10 fg DNA template of S. aureus per reaction, with no detectable false-positive response, which was 103-fold more sensitive than previously reported LAMP assay. The assay was shown to detect two strains of S. aureus, and did not detect 24 non-S. aureus strains, providing improved specificity over previously reported LAMP assays.