Sequence-specific methods for homogeneous, real-time detection of lamp products

Abstract

Presented herein are methods and compositions for generating sequence-specific, secondary amplification products during Loop-mediated Isothermal Amplification (LAMP). Conventional LAMP produces a preponderance of high molecular weight DNA structures concatenated into self-complementary hairpins, which are not amenable to detection by routine probe-based hybridization methods, making multiplex detection of two or more targets or sequence variants in closed-tube formats extremely difficult. Provided herein, for example, are methods for generating secondary LAMP products bearing a fragment of the original target sequence embedded within low-molecular weight products that are devoid of competitive hairpin structures, the lack of which enhances probe-based detection of target sequences. These secondary products can, for example, be produced in real-time, during the LAMP process, and can provide the option of detecting multiple target sequences within a single tube using, e.g., a homogenous, real-time fluorescence format.