A fast, accurate strategy based on LAMP-NALF for detecting human adenovirus serotype 3 in children

Objective: To establish a rapid, convenient and accurate visual detection protocol for human adenovirus serotype 3 in children's respiratory infections based on LAMP-NALF. Methods: Adenovirus serotypes prevalent in the region (Changzhou City, Jiangsu Province, China) were analyzed by assessing the results of tNGS analysis of respiratory pathogens in children. On this basis, multiple sets of LAMP primers were designed for the adenovirus hexon gene as the detection target. Optimal primer combinations were further screened, and the composition of the reagent system and amplification procedures were improved to achieve efficient amplification. To realize an instrument-independent, fast-testing strategy, by combining NALF, which is based on the principle of lateral chromatography, small molecule antigens were modified for primers. Moreover, amplification products were bound to the corresponding antibodies on nucleic acid chromatography test strips for visualization. Finally, a series of tests were performed to validate the sensitivity, specificity, and methodological consistency of the LAMP-NALF assay. Results: Adenovirus infections in children in this region during this period were mainly caused by serotype 3. The optimal amplification temperature for optimal primers of LAMP was 65 °C. The combination of LAMP and NALF could detect femtogram-grade positive plasmid of the hexon gene; besides, there was no cross-reactivity with five other types of common respiratory pathogens in children. The agreement between the LAMP-NALF assay and tNGS/qPCR for detecting the adenovirus in 100 respiratory clinical specimens was 98%. Conclusion: The combination of LAMP and NALF can achieve rapid, convenient and accurate detection of human adenovirus type 3, which can provide a powerful support for the pathogenic diagnosis of adenoviral infections in children, thus contributing to the promotion of the development of point-of-care testing for pathogens.