Isothermal Nucleic Acid Amplification-Based Lateral Flow Testing for the Detection of Plant Viruses

Abstract

Isothermal nucleic acid amplification-based lateral flow testing (INAA-LFT) has emerged as a robust technique for on-site pathogen detection, providing a visible indication of pathogen nucleic acid amplification that rivals or even surpasses the sensitivity of real-time quantitative PCR. The isothermal nature of INAA-LFT ensures consistent conditions for nucleic acid amplification, establishing it as a crucial technology for rapid on-site pathogen detection. However, despite its considerable promise, the widespread application of isothermal INAA amplification-based lateral flow testing faces several challenges. This review provides an overview of the INAA-LFT procedure, highlighting its advancements in detecting plant viruses. Moreover, the review underscores the imperative of addressing the existing limitations and emphasizes ongoing research efforts dedicated to enhancing the applicability and performance of this technology in the realm of rapid on-site testing.

Keywords: LAMP; RPA; detection; isothermal; lateral flow; on-site; plant virus.

Figure 1

Workflow of the INAA-LFT. Total DNA or RNA is extracted from the leaves of virus-infected plants, and then used as template for INAA. The labeled target nucleic acid fragments are visualized on a paper-based strip. A control line is also present on the strip to verify the correct functioning of the test.

Figure 2

Optimized workflow of INAA-LFT at present, and its future trajectory in the detection of plant virus. In the current INAA-LFT system, starting with sampling, there are three subsequent processes, including reaction preparation, isothermal amplification, and product dilution and detection, which take a total of 20–60 min. In the future, if the detection time can be reduced to 8–10 min, sample processing, reaction, and detection can be completed using one device, and can be combined with artificial intelligence (AI) to achieve standardization (parts framed by yellow lines). This will open new avenues for improving the overall performance and applicability of this technology in diverse settings.