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. 2022 Aug 24:10:964244.
doi: 10.3389/fbioe.2022.964244. eCollection 2022.

Novel miniaturized fluorescence loop-mediated isothermal amplification detection system for rapid on-site virus detection

Yanqi Wu  1   2 , Liping Bai  1 , Chengfu Ye  2 , Yuhong Guan  2 , Kunming Yan  2 , Hui Chen  3 , Zhihong Jiang  1
Affiliations

Novel miniaturized fluorescence loop-mediated isothermal amplification detection system for rapid on-site virus detection

Yanqi Wu et al. Front Bioeng Biotechnol. .

Abstract

New pathogen outbreaks have progressed rapidly and are highly infectious in recent years, increasing the urgency of rapid and accurate detection of pathogenic microorganisms. Based on the point-of-care testing (POCT) requirements, in this study, a real-time fluorescent loop-mediated isothermal amplification (LAMP) detection system was developed and applied to pathogen detection. The system is compact and portable, with good uniformity and reproducibility, and it can detect pathogens rapidly and effectively. For norovirus detection, the linear range was 100-106 copies/μL. The system can achieve the theoretical sensitivity of LAMP detection, conclusions could be obtained within 35 min, and quantitative detection was possible. The test results of 45 clinical samples were consistent with quantitative PCR (qPCR) and clinical results, and the accuracy could reach 100%. This system has the characteristics of portability, speed, and POCT accuracy, and the cost is much lower than that of commercial qPCR. Therefore, it is suitable for remote areas or places with relatively poor conditions and environments requiring on-site conditions. It can also be widely used to detect various epidemics and unexpected diseases.

Keywords: LAMP; POCT; aerosol pollution; fluorescence detection; norovirus.

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Conflict of interest statement

Authors YW, CY, YG, and KY were employed by the company Shenzhen LemnisCare Medical Technology Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
(A) Overall schematic of the system; (B). Schematic of the heat trap and fluorescence modules; (C). Multi-channel fluorescence signal acquisition curve.
FIGURE 2
FIGURE 2
16-well temperature uniformity curve.
FIGURE 3
FIGURE 3
Six-well uniformity test results: (A) Our system. (B) Commercial qPCR instrument.
FIGURE 4
FIGURE 4
Results of norovirus sensitivity. (A) LAMP amplification curves of six concentration gradients in the system; (B) Standard curve for LAMP assay; (C) qPCR amplification curves of six concentration gradients in the system; (D) Standard curve for qPCR assay.
FIGURE 5
FIGURE 5
Results of the three LAMP experiments with high, medium, and low concentrations. (A–C) show the three LAMP amplification curves, respectively; (D). Stability of the three LAMP experiments.
FIGURE 6
FIGURE 6
Results of clinical samples by RT-LAMP and RT-qPCR. (A) RT-LAMP on our system; (B) RT-qPCR on the commercial instrument.
See this image and copyright information in PMC

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