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

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 14 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.

Visual Detection of Isothermal Nucleic Acid Amplification Using pH-Sensitive Dyes

“…), or 10 pg100 ng (as indicated) HeLa genomic DNA (New England Biolabs). Bst DNA polymerase, Large Fragment or Bst 2.0 DNA polymerase (New England Biolabs) at 0.32 U/L proved effective, but all displayed data were obtained using Bst 2.0 DNA polymerase because of its more rapid amplification times (23). Both standard (8 U/L stock) and high (120 U/L) polymerase concentrations were effective in pH-detected reactions, but all colorimetric data shown were obtained using the higher concentration enzyme to minimize buffer carryover (i.e., 26 M Tris vs. 400 M).…”

“…All solutions of primers, dNTPs, and template DNAs were prepared in water to minimize buffer carryover (except HeLa DNA 0.1 mg/mL stock for the highest template input). No betaine was added to the reaction buffer because it was not beneficial to amplification of the targets used here (23,24). All reactions were repeated at least once and were typically performed in triplicate.…”

Electricity-Free Amplification and Detection for Molecular Point-of-Care Diagnosis of HIV-1

“…Other studies have also noted decreased single assay performance in biplexed LAMP reactions, with one study demonstrating a negative effect on time to result [47]. In this study, since the β-actin mRNA and DNA were more concentrated than the HIV-1 RNA, they may amplify more effectively and, therefore, restrict the HIV-1 assays’ sensitivity by sequestering the limited reagents for reverse transcription and DNA amplification.…”

“…Multiplexed assays are commonly used in PCR for the detection of an internal control to confirm absence of inhibition. However, only a limited number of studies have examined multiplexed LAMP, [45]–[47] and to our knowledge, none have used NALF as the detection method. To demonstrate the utility of this evolving platform, we paired the technology with a NALF-detection cassette and evaluated the performance of the combined components using a biplexed LAMP assay for the detection of HIV-1 and a ß-actin internal control.…”

Expanding the MDx toolbox for filarial diagnosis and surveillance

“…In the future, a lateral flow dipstick or ELISA based on hybridization of LAMP products to specific immobilized probes and detection of the captured amplicons by immunoassay technology is feasible [68]. Various multiplexing options have been described including those in a single reaction [69] and on a disposable microfluidic chip [70]. A digital LAMP on a sample self-digitization chip that requires less than 2 ml of sample volume is also available [71].…”