Locked Nucleic Acid and Minor Groove Binder Probes Improve the Specificity of Isothermal Amplification Detection

Isothermal amplification reactions have diverse applications in molecular diagnostics, point-of-care detection, and synthesis of nucleic acids. Isothermal amplification techniques eliminate the need for temperature cycling and the related equipment. However, a common issue of isothermal amplification is the formation of nonspecific amplification byproducts that cause false-positive detection. We report here the development and design principles of sequence-specific minor groove binder (MGB) and locked nucleic acid (LNA) probes, which significantly improve the detection specificity. We show an example of integrating loop-mediated isothermal amplification (LAMP) with the MGB and LNA probes and demonstrate an application of the integrated method to the specific detection of Streptococcus pyogenes (group A streptococcus [GAS]) and skin and wound specimens. The technique reduced background and eliminated false positives generated by nonspecific amplification byproducts. The method achieved a detection limit of 25 copies of the target speB DNA with a 30 min LAMP-probe reaction. Results from the analysis of 171 skin and wound specimens were consistent with clinical diagnostics of GAS infections, confirming 100% positive predictive value (PPV) and 100% negative predictive value (NPV) of the assay. The modular design of LAMP and the MGB and LNA detection probes can be readily adapted for the detection of other nucleic acid targets.