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

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.