Multiplex reverse transcription loop-mediated isothermal amplification combined with nanoparticle-based lateral flow biosensor for the diagnosis of COVID-19

Biosens Bioelectron. 2020 Oct 15;166:112437. doi: 10.1016/j.bios.2020.112437. Epub 2020 Jul 15.


The ongoing global pandemic (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a huge public health issue. Hence, we devised a multiplex reverse transcription loop-mediated isothermal amplification (mRT-LAMP) coupled with a nanoparticle-based lateral flow biosensor (LFB) assay (mRT-LAMP-LFB) for diagnosing COVID-19. Using two LAMP primer sets, the ORF1ab (opening reading frame 1a/b) and N (nucleoprotein) genes of SARS-CoV-2 were simultaneously amplified in a single-tube reaction, and detected with the diagnosis results easily interpreted by LFB. In presence of FITC (fluorescein)-/digoxin- and biotin-labeled primers, mRT-LAMP produced numerous FITC-/digoxin- and biotin-attached duplex amplicons, which were determined by LFB through immunoreactions (FITC/digoxin on the duplex and anti-FITC/digoxin on the test line of LFB) and biotin/treptavidin interaction (biotin on the duplex and strptavidin on the polymerase nanoparticle). The accumulation of nanoparticles leaded a characteristic crimson band, enabling multiplex analysis of ORF1ab and N gene without instrumentation. The limit of detection (LoD) of COVID-19 mRT-LAMP-LFB was 12 copies (for each detection target) per reaction, and no cross-reactivity was generated from non-SARS-CoV-2 templates. The analytical sensitivity of SARS-CoV-2 was 100% (33/33 oropharynx swab samples collected from COVID-19 patients), and the assay's specificity was also 100% (96/96 oropharynx swab samples collected from non-COVID-19 patients). The total diagnostic test can be completed within 1 h from sample collection to result interpretation. In sum, the COVID-19 mRT-LAMP-LFB assay is a promising tool for diagnosing SARS-CoV-2 infections in frontline public health field and clinical laboratories, especially from resource-poor regions.

Keywords: COVID-19; Lateral flow biosensor; Multiplex reverse transcription loop-mediated isothermal amplification; Rapid diagnosis; SARS-CoV-2.

Publication types

  • Validation Study

MeSH terms

  • Betacoronavirus / genetics*
  • Betacoronavirus / isolation & purification*
  • Biosensing Techniques* / instrumentation
  • Biosensing Techniques* / methods
  • Biosensing Techniques* / statistics & numerical data
  • COVID-19
  • COVID-19 Testing
  • China / epidemiology
  • Clinical Laboratory Techniques* / instrumentation
  • Clinical Laboratory Techniques* / methods
  • Clinical Laboratory Techniques* / statistics & numerical data
  • Coronavirus Infections / diagnosis*
  • Coronavirus Infections / epidemiology
  • Coronavirus Infections / virology*
  • Equipment Design
  • Feasibility Studies
  • Humans
  • Limit of Detection
  • Molecular Diagnostic Techniques
  • Multiplex Polymerase Chain Reaction / methods
  • Multiplex Polymerase Chain Reaction / statistics & numerical data
  • Nanoparticles
  • Nanotechnology
  • Nucleic Acid Amplification Techniques
  • Pandemics*
  • Pneumonia, Viral / diagnosis*
  • Pneumonia, Viral / epidemiology
  • Pneumonia, Viral / virology*
  • RNA, Viral / analysis
  • RNA, Viral / genetics
  • SARS-CoV-2
  • Sensitivity and Specificity


  • RNA, Viral

Supplementary concepts

  • LAMP assay