Visual Detection of Single-Nucleotide Polymorphisms Using Molecular Beacon Loop-Mediated Isothermal Amplification with Centrifuge-Free DNA Extraction

Anal Chem. 2019 Jun 4;91(11):6991-6995. doi: 10.1021/acs.analchem.9b01762. Epub 2019 May 23.

Abstract

Loop-mediated isothermal amplification (LAMP) is a powerful nucleic acid amplification technique due to its rapid and sensitive nature. These characteristics, in addition to low-cost and robustness, make LAMP an attractive alternative to polymerase chain reaction for point-of-care applications. However, sequence-specific detection remains a formidable challenge, particularly when single-nucleotide resolution is required. In this study, a LAMP method is developed for facile visual detection of single-nucleotide polymorphisms (SNPs) using molecular beacons (MBs) by exploiting the dual-fluorescence of fluorescein (6-FAM) and hydoxynaphthol blue (HNB). The method is coupled with solid-phase microextraction (SPME) to facilitate rapid extraction and detection of the target sequence. This work expands the use of MBs in LAMP for the visual detection of SNPs and facilitates the development of future LAMP assays for a wide-range of targets.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Centrifugation
  • DNA / isolation & purification*
  • Escherichia coli K12 / genetics
  • Fluorescein / chemistry
  • Fluorescent Dyes / chemistry
  • Nucleic Acid Amplification Techniques / methods*
  • Polymorphism, Single Nucleotide*
  • Solid Phase Microextraction
  • Spectrometry, Fluorescence

Substances

  • Fluorescent Dyes
  • DNA
  • Fluorescein