Development of Dual 'RT-LAMP-LFA' Rapid Detection Technology With Gold Magnetic Nanoparticles for Influenza Virus

Abstract

Seasonal and persistent outbreaks of influenza viruses represent a significant challenge to global public health. Rapid, convenient and accurate diagnosis methods of influenza viruses are crucial for timely treatment to mitigate morbidity and mortality during both seasonal epidemics and pandemics. However, current diagnostic tools often face limitations in speed, accuracy or complexity of result interpretation; there is a great need for more efficient detection technology for influenza virus, especially for use in resource-limited settings or during large-scale outbreaks. This study developed a dual 'RT-LAMP-LFA' detection technology with gold magnetic nanoparticles for influenza virus. This method can simultaneously detect influenza A and B genes as well as internal reference genes within 35 min, with a detection limit of 80 copies/mL. This is the first time the RNase P gene has been introduced into a gold magnetic nanoparticle lateral flow assay system as a quality control measure to monitor the entire sampling and amplification process in virus detection and reveals the effects of loop primer deficiencies on the stability of the dual 'RT-LAMP-LFA' detection technology. Using fluorescent PCR detection technology as a benchmark, the analysis of a total of 70 clinical samples demonstrated a 100% agreement rate, confirming the applicability and accuracy of the dual 'RT-LAMP-LFA' detection system. This dual 'RT-LAMP-LFA' detection technology offers a novel option for diagnostic technology in hierarchical medical testing, presenting significant social importance and broad application prospects.

Keywords: dual loop‐mediated isothermal amplification; gold magnetic nanoparticles; influenza virus; lateral flow assay.

FIGURE 1

The Principle of the Dual ‘RT‐LAMP‐LFA’ Detection Technology. (a) The dual ‘RT‐LAMP‐LFA’ detection technology. (b) Design and visualisation principles of the LFA system. (1) Sample pad; (2) conjugated pad; (3) nitrocellulose membrane (NC membrane); (4) absorbent pad; and (5) plastic cushion. (Created with BioRender.com.)

FIGURE 2

Assessment of the reacting conditions of the primer sets. (a) The temperature assessment of the FluA gene primer set, FluB gene primer set and internal reference gene primer set, respectively. (b) The specificity of the primer set (Lane M: DNA marker; Lane 1: FluA gene primer set + internal reference gene template; Lane 2: FluA gene template + internal reference gene primer set; Lane 3: FluB gene primer set + internal reference gene template; and Lane 4: FluB gene template + internal reference gene primer set). (c) The reaction time of different primer ratios. (d) The reaction time of different primer concentrations. (The reaction time is shown by Genie II; error bars represent the mean ± standard deviation (SD) of three technical replicates.)

FIGURE 3

Analysis of loop primers affecting the amplification of different targets. (a) The effect of primer concentration of reference gene rings on the dual ‘RT‐LAMP‐LFA’ detection technique of FluA was analysed by the LFA system, heatmap and bar chart. (b) The effect of primer concentration of reference gene rings on the dual ‘RT‐LAMP‐LFA’ detection technique of FluB was analysed by the LFA system, heatmap and bar chart. (Heat maps and bar charts represent the intensities of the C and T lines analysed by Image J, respectively, and error bars represent the mean ± standard deviation (SD) of three technical replicates.)

FIGURE 4

Performance evaluation of the dual ‘RT‐LAMP‐LFA’ detection technology. (a, d) Specific analysis of FluA and FluB by the dual ‘RT‐LAMP‐LFA’ assay. (b, e) Sensitivity analysis of FluA and FluB with the dual ‘RT‐LAMP‐LFA’ assay for different copy numbers. (c, f) Instrument suitability analysis of FluA and FluB by the dual ‘RT‐LAMP‐LFA’ assay. Figure (a–f) shows the intensity of the C and T lines analysed by Image J, respectively, and the error bars represent the mean ± standard deviation (SD) of the three technical replicates.

FIGURE 5

Clinical application of dual ‘RT‐LAMP‐LFA’ detection technology. (a) Schematic diagram of the dual ‘RT‐LAMP‐LFA’ test results in some clinical samples. (b) Heat map analysis shows the results of 70 clinical samples tested using dual ‘RT‐LAMP‐LFA’ detection technology, and the signal value expressed by the heat map is the T‐line signal intensity of clinical samples analysed by Image J. (c) Analysis of C line and T line of 70 nasopharyngeal swab samples showed the signal of C line value of all samples, T line value of positive samples and T line value of negative samples. (d) Correlation between RT‐LAMP‐LFA and RT‐PCR in 70 clinical samples. Signal intensities of the C and T lines were analysed by Image J.