Development and validation of a multi-platform LAMP system for rapid HAdV-3 and HAdV-7 detection

Abstract

Purpose: We developed a loop-mediated isothermal amplification (LAMP) multi-platform detection system that overcomes the limitations of traditional polymerase chain reaction (PCR) in timeliness and equipment dependency, establishing a rapid, accurate diagnostic framework for Human adenovirus types 3 and 7 (HAdV-3 and HAdV-7) across resource-varied clinical settings.

Methods: LAMP primers were designed based on the conserved regions of the Hexon genes of HAdV-3 and HAdV-7. The calcein, immunochromatography (IC), and fluorescence probe methods were used. Sensitivity and specificity analyses determined each method's performance. Additionally, 188 clinical samples were analyzed in parallel using a commercial PCR kit.

Results: The calcein and IC methods achieved a limit of detection (LOD) of 2.5 copies/reaction. The fluorescent probe method demonstrated superior sensitivity, with an LOD of 1 copy/reaction and a median Ct value of 7.3, 72.8% lower than that of qPCR (median Ct 26.9; p < 0.05). All three platforms exhibited 100% specificity, with no cross-reactivity observed against SARS-CoV-2 or other tested respiratory pathogens. Clinical validation showed 100% concordance between the fluorescent probe LAMP assay and qPCR (κ = 1.00; 95% CI: 1.00-1.00). The actual detection time was ≤ 20 min, and the assay performed reliably in low-viral-load and co-infection cases.

Conclusion: The multi-platform LAMP system established in this study has created a hierarchical detection network characterized as "preliminary screening-quantitative", specifically designed to meet the diverse needs of grassroots, field, and laboratory settings. This system offers efficient multi-scenario solutions for the prevention and control of respiratory infections.

Keywords: Calcein; HAdV; Immunochromatography; LAMP; Respiratory infections.

Fig. 1

Position of primers in human adenovirus sequences

Fig. 2

Sensitivity analysis of three LAMP detection methods. (plasmid concentrations: 1 × 10⁶ to 1 × 10⁰ copies/mL). a calcein method (fluorescence detection at 302 nm excitation), b calcein method (visual inspection), c IC method, and d fluorescent probe method. Lanes 1–7: Plasmid DNA concentrations (1 × 10⁶, 1 × 10⁵, 1 × 10⁴, 1 × 10³, 1 × 10², 1 × 10¹, 1 × 10⁰ copies/mL). LAMP, loop-mediated isothermal amplification; IC, immunochromatography; NC, negative control; C, control line; T, test line

Fig. 3

Sensitivity analysis of three LAMP detection methods. (plasmid concentrations: 1000, 500, 200, 100 copies/mL). a calcein method (fluorescence detection at 302 nm excitation), b calcein method (visual inspection), c IC method, and d fluorescent probe method. Lanes 1–4: Plasmid DNA concentrations (1000, 500, 200, and 100 copies/mL). LAMP, loop-mediated isothermal amplification; IC, immunochromatography; NC, negative control; C, control line; T, test line

Fig. 4

Specificity analyses of three LAMP detection methods. a calcein method (fluorescence detection at 302 nm excitation), b calcein method (visual inspection), c IC method, d fluorescent probe method. Lanes 1–11: common respiratory pathogen standards, including HAdV-7, IAV H1N1, IAV H3N2, IAV H7N9, IBV Victoria, IBV Yamagata, SARS-CoV-2, PIV 1, HRSV A, HRSV B, and rhinovirus. LAMP, loop-mediated isothermal amplification; IC, immunochromatography; NC, negative control; PC, positive control; C, control line; T, test line

Fig. 5

A comparison of the consistency between LAMP and qPCR methods in detecting 188 clinical samples. a LAMP results (85 positive samples, 9 negative samples), b LAMP results (94 negative samples), c qPCR results [corresponding to (a)], and d qPCR results [corresponding to (b)]. LAMP, loop-mediated isothermal amplification; PCR, polymerase chain reaction; NC, negative control; PC, positive control