Detection of Reverse Transcriptase LAMP-Amplified Nucleic Acid from Oropharyngeal Viral Swab Samples Using Biotinylated DNA Probes through a Lateral Flow Assay

Biosensors (Basel). 2023 Nov 17;13(11):988. doi: 10.3390/bios13110988.

Abstract

This study focuses on three key aspects: (a) crude throat swab samples in a viral transport medium (VTM) as templates for RT-LAMP reactions; (b) a biotinylated DNA probe with enhanced specificity for LFA readouts; and (c) a digital semi-quantification of LFA readouts. Throat swab samples from SARS-CoV-2 positive and negative patients were used in their crude (no cleaning or pre-treatment) forms for the RT-LAMP reaction. The samples were heat-inactivated but not treated for any kind of nucleic acid extraction or purification. The RT-LAMP (20 min processing time) product was read out by an LFA approach using two labels: FITC and biotin. FITC was enzymatically incorporated into the RT-LAMP amplicon with the LF-LAMP primer, and biotin was introduced using biotinylated DNA probes, specifically for the amplicon region after RT-LAMP amplification. This assay setup with biotinylated DNA probe-based LFA readouts of the RT-LAMP amplicon was 98.11% sensitive and 96.15% specific. The LFA result was further analysed by a smartphone-based IVD device, wherein the T-line intensity was recorded. The LFA T-line intensity was then correlated with the qRT-PCR Ct value of the positive swab samples. A digital semi-quantification of RT-LAMP-LFA was reported with a correlation coefficient of R2 = 0.702. The overall RT-LAMP-LFA assay time was recorded to be 35 min with a LoD of three RNA copies/µL (Ct-33). With these three advancements, the nucleic acid testing-point of care technique (NAT-POCT) is exemplified as a versatile biosensor platform with great potential and applicability for the detection of pathogens without the need for sample storage, transportation, or pre-processing.

Keywords: LFA; NAAT-LFA; RT-LAMP; semi-quantitative; surveillance-based diagnostics.

MeSH terms

  • Biotin
  • COVID-19* / diagnosis
  • DNA
  • DNA Probes
  • Fluorescein-5-isothiocyanate
  • Humans
  • Molecular Diagnostic Techniques / methods
  • Nucleic Acid Amplification Techniques / methods
  • Nucleic Acids*
  • RNA, Viral / genetics
  • RNA-Directed DNA Polymerase / genetics
  • SARS-CoV-2 / genetics
  • Sensitivity and Specificity

Substances

  • RNA-Directed DNA Polymerase
  • Nucleic Acids
  • Biotin
  • Fluorescein-5-isothiocyanate
  • DNA
  • RNA, Viral
  • DNA Probes