Simultaneous Multiple Target Detection in Real-Time Loop-Mediated Isothermal Amplification

TLDR

Loop‑mediated isothermal amplification (LAMP) is a rapid, reliable nucleic‑acid amplification method, yet real‑time detection has been limited to single targets. The authors designed LAMP primers with a quencher‑fluorophore duplex that releases fluorescence upon strand separation, enabling multiplex real‑time detection and expanding LAMP’s diagnostic utility. This duplex‑modified primer system allows simultaneous detection of 1–4 target sequences in a single reaction tube using a standard real‑time fluorimeter. The approach is highly reproducible and sensitive (detecting <100 copies), covers a 7‑order dynamic range, and with Bst 2.0 or Bst 2.0 WarmStart polymerases achieves 50 % faster amplification signals.

Abstract

Loop-mediated isothermal amplification (LAMP) is a rapid and reliable sequence-specific isothermal nucleic acid amplification technique. To date, all reported real-time detection methods for LAMP have been restricted to single targets, limiting the utility of this technique. Here, we adapted standard LAMP primers to contain a quencher-fluorophore duplex region that upon strand separation results in a gain of fluorescent signal. This approach permitted the real-time detection of 1–4 target sequences in a single LAMP reaction tube utilizing a standard real-time fluorimeter. The methodology was highly reproducible and sensitive, detecting below 100 copies of human genomic DNA. It was also robust, with a 7-order of magnitude dynamic range of detectable targets. Furthermore, using a new strand-displacing DNA polymerase or its warm-start version, Bst 2.0 or Bst 2.0 WarmStart DNA polymerases, resulted in 50% faster amplification signals than wild-type Bst DNA polymerase, large fragment in this new multiplex LAMP procedure. The coupling of this new multiplex technique with next generation isothermal DNA polymerases should increase the utility of the LAMP method for molecular diagnostics.