Loop-mediated isothermal amplification combined with lateral flow biosensor for rapid and sensitive detection of monkeypox virus

Abstract

The ongoing outbreak of the monkeypox, caused by monkeypox virus (MPXV), has been a public health emergency of international concern, indicating an urgent need for rapid and sensitive MPXV detection. Here, we designed a diagnostic test based on loop-mediated isothermal amplification (LAMP) and nanoparticle-based lateral flow biosensor(LFB)for diagnosis of MPXV infection, termed MPX-LAMP-LFB. A set of six LAMP primers was designed based the ATI gene of MPXV, and LAMP amplification of MPXV templates was performed at 63°C for only 40 min. The results were rapidly and visually decided using the LFB test within 2 min. The MPX-LAMP-LFB assay can specifically detect MPXV strains without cross-reaction with non-MPXV pathogens. The sensitivity of the MPX-LAMP-LFB assay is as low as 5 copies/μl of plasmid template and 12.5 copies/μl of pseudovirus in human blood samples. The whole process of the MPX-LAMP-LFB assay could be completed ~1 h, including rapid template preparation (15 min), LAMP reaction (40 min)and result reporting (<2 min). Collectively, MPX-LAMP-LFB assay developed here is a useful tool for rapid and reliable diagnosis of MPXV infection.

Keywords: diagnosis; lateral flow biosensor; loop-mediated isothermal amplification; monkeypox virus; nanoparticles.

Figure 1

Primer sequences and locations used in this study. Right and left arrows show sense and complementary sequences, respectively. The colored text indicates the position of primers, including two outer primers (F3 and B3), two inner primers (FIP and BIP) and two loop primers(LF and LB).

Figure 2

Effectiveness of the primer set for the MPX-LAMP-LFB assay. The effectiveness of the primer set for the MPX-LAMP-LFB assay was verified by testing the LAMP products with real-time turbidity (A), visual detection reagent (B), and LFB (C) methods. Curve/Tube/Biosensor 1, the ATI-plasmid that used as positive control, which was effectively amplified with LAMP reaction at 65°C; Curve/Tube/Biosensor2, the genomic DNA of influenza virus A that used as negative control; Curve/Tube/Biosensor3, the blank control (DW).

Figure 3

Specificity conformation for MPX–LAMP-LFB assay. Specificity of the MPX-LAMP-LFB assay was confirmed by testing the LAMP products with visual detection reagent (A) and LFB (B) methods. Biosensors/Tubes 1–2 showed the LAMP reaction results of ATI-plasmid and the pseudotyped virus; Biosensors/Tubes 3–19 showed the LAMP reaction results of the 17 non-MPXV viral pathogens (Supplementary Table S1); Biosensor/Tube 20 showed the LAMP reaction results of blank control. TL, test line; CL, control line.

Figure 4

Sensitivity confirmation of MPX–LAMP-LFB assay. The sensitivity of the MPX–LAMP-LFB assay was assessed by using the serially diluted ATI-plasmid (A) and the constructed pseudotyped virus (B). Three monitoring formats, including turbidity (up row), visual detection reagent (middle row), and LFB (bottom row), were applied to detect LAMP products. Templates 1–8 in A were the ATI-plasmid with concentrations of 5 × 10⁵, 5 × 10⁴, 5 × 10³, 5 × 10², 5 × 10¹, 5 × 10⁰, and 5 × 10⁻¹copies permicroliter and the uncontaminated blood sample; Templates 1–8 in B were the constructed pseudotyped virus with concentrations of 1.25 × 10³, 1.25 × 10², 1.25 × 10¹, 1.25 × 10⁰, 1.25 × 10⁻¹, 1.25 × 10⁻², and 1.25 × 10⁻³copies per microliter and the uncontaminated lesional swab sample. TL, test line; CL, control line.

Figure 5

Clinical feasibility confirmation of the MPX-LAMP-LFB assay in simulated blood and lesional swab specimens. Clinical feasibility of the MPX-LAMP-LFB assay was confirmed by using the serially diluted simulated blood specimens with nucleic acid extraction process (A) and without nucleic acid extraction process (B), and the serially diluted simulated lesional swab specimens with nucleic acid extraction process (C) and without nucleic acid extraction process (D). All the specimens were detected using turbidity (top row), visual detection reagent (middle row)and LFB (bottom row). Curves/Tubes/Biosensors 1–8 represented the amplification results of the pseudotyped virus levels in blood samples of 1.25 × 10³, 1.25 × 10², 1.25 × 10¹, 1.25 × 10⁰, 1.25 × 10⁻¹, 1.25 × 10⁻², and 1.25 × 10⁻³copies permicroliter and DW.TL, test line; CL, control line.