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

Abstract

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.

Loop-Mediated Isothermal Amplification (LAMP): A Rapid, Sensitive, Specific, and Cost-Effective Point-of-Care Test for Coronaviruses in the Context of COVID-19 Pandemic

“…Recent investigations focus on simplifying the procedure even further by incorporating everything in a 'one-step' or 'single-tube' assay using nanoparticle-based biosensors [81] or by including a magnetic bead capture step during processing of dry swabs to maximize viral RNA yield [81,82]. Although such advances are highly promising, further optimization studies are required to improve sensitivity and specificity.…”

Pharmaceutical nanotechnology: which products are been designed against COVID-19?

“…This dual-functional plasmid biosensor combines the plasmatic photothermal effect with localized plasmon resonance transduction, showing high sensitivity to SARS-CoV-2 with a lower detection limit up to a concentration of 0.22 pM, in addition to allowing accurate detection in a multigenic mixture. Zhu et al (2020) proposed a diagnostic technique for COVID-19 using nanoparticles, to which they created an isothermal amplification mediated by a multiplex reverse transcription loop coupled to a nanoparticlebased lateral flow biosensor. The results showed that the analytical technique showed a sensitivity of 100% for SARS-CoV-2, after the analysis of 33 oropharyngeal samples from patients with COVID-19.…”

“Tomorrow Never Dies”: Recent Advances in Diagnosis, Treatment, and Prevention Modalities against Coronavirus (COVID-19) amid Controversies

“…Among these, loop-mediated isothermal amplification (LAMP) has been implied successfully for coronavirus detection [42][43][44][45]. LAMP technique can amplify target nucleic acid sequence using two or three sets of primers and a polymerase at a constant temperature (~60-65 • C) [46][47][48]. In comparison to PCR-based technique, LAMP can produce considerably higher amount of DNA with high strand displacement and replication activity due to the use of additional pair of "loop primers".…”

“…The sensitivity of this portable method is equivalent to a commercial RT-qPCR test with only heating and visual inspection. Zhu et al [47] demonstrated a successful and accurate diagnosis of COVID-19 using one-step RT-LAMP coupled with nanoparticles-based biosensor (NBS) assay (RT-LAMP-NBS) within approximately 1 h (from sample collection to result interpretation). They have employed two designed LAMP primer sets (F1ab-RT-LAMP and np-RT-LAMP), heating block (to maintain a constant temperature at 63 • C), a real-time turbidity (LA-320C) and visual detection reagents (VDR) in addition to NBS interpretation to simultaneously amplify and detect genes of SARS-CoV-2 in a "one-step" and "single-tube" reaction.…”