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.

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

Fig. 1

Outline of COVID-19 RT-LAMP-LFB design. A. Outline of LAMP assay with LF* and LB*.Top row, outline of LAMP with LF* and LB*; Bottom row, schematic depiction of the new forward/backward loop primers (LF*/LB*). LF* was labeled with a hapten at the 5′ end, and LB* was labeled with biotin at the 5′ end. B. Mechanistic description of the COVID-19 RT-LAMP-LFB assay.(1), Preparing the amplification mixtures. (2), RT-LAMP reaction. (3), The detectable COVID-19 RT-LAMP products after reaction. ORF1ab-RT-LAMP products were simultaneously labeled with FITC and biotin, and the N-RT-LAMP products were labeled with Dig and biotin. C, The principle of LFB for visualization of COVID-19 RT-LAMP products. (1), The details of LFB. (2), The principle of LFB for COVID-19 RT-LAMP products. (3), Interpretation of the COVID-19 RT-LAMP results. I, a positive result for ORF1ab and N (Test line 1, Test line 2 and Control line appear on the LFB); II, a positive result for N (Test line 2 and Control line appear on the detection region); III, a positive result for ORF1ab (Test line 1 and Control line appear on the detection region); IV, negative (only the control line appears on the LFB). *Note, LFB I (Positive result for ORF1ab and N genes), LFB II (Positive for N gene) and LFB III (Positive result for ORF1ab gene) can be judged as positive for SARS-CoV-2. D, Primer design of COVID-19 mRT-MCDA-LFB assay. Up row, SARS-CoV-2 genome organization (GenBank: MN908947, Wuhan-Hu-1). The length of all genes are not displayed to scale. Bottom row, nucleotide sequence and location of ORF1ab and N gene used to design COVID-19 RT-LAMP primers. Part of the nucleotide sequences of ORF1ab (Left) and N (Right) are listed. The sites of primer sequence are underlined. Right arrows and Left arrows show the sense and complementary sequences that are used. * Note: ORF1ab (Open reading frame 1a/b); S (Spike protein); E (Envelope protein); M (Membrane protein); N (Nucleoprotein); Accessory proteins (3, 6, 7a, 7b, and 9b).

Fig. 2

The workflow of COVID-19 mRT-LAMP-LFB assay. Four steps, including sample collection (3 min), rapid RNA extraction (15 min), mRT-LAMP reaction (40 min) and result reporting (<2 min), were required for conduct the COVID-19 RT-LAMP-LFB diagnosis test, and the whole process could be completed within 60 min.

Fig. 3

Sensitivity of COVID-19 mRT-LAMP-LFB assay. A, LFB applied for reporting the results; B, Real-time turbidity applied for reporting the results; C, Visual detection reagent applied for reporting the results. LFB (A)/Signals (B)/Tubes (C) 1–8 represented the plasmid levels (each of ORF1ab-plasmid and N-plasmid) of 1.2 × 10⁴, 1.2 × 10³, 1.2 × 10², 1.2 × 10¹, 1.2 × 10⁰, 1.2 × 10⁻¹, 1.2 × 10⁻² copies per reaction and blank control (DW). The plasmid levels of 1.2 × 10⁴ to 1.2 × 10¹ copies per reaction produced positive reactions.

Fig. 4

COVID-19 mRT-LAMP-LFB results on 33 COVID-19 infected patient samples. Results were analyzed in accordance with the guidance displayed in Fig. S1. 33 samples (S1 to S33) were collected from 33 patients. PC (Positive control), 1.2 × 10² copies each of ORF1ab-plasmids and N-plasmids were added into COVID-19 mRT-LAMP reaction mixture. NTC (Negative control) and BC (Blank control), RNA template of H1N1 and distilled water were added into COVID-19 mRT-LAMP reaction mixtures, respectively.