A comprehensive guide to loop-mediated isothermal amplification, an emerging diagnostic tool for plant pathogenic fungi

Abstract

The detection and identification of plant pathogenic fungi are crucial for effective plant protection measures. In the past two decades, loop-mediated isothermal amplification (LAMP) has emerged as a simple and cost-efficient tool for plant disease diagnosis, overcoming many drawbacks of traditional and PCR-based methods. LAMP relies on efficient DNA synthesis at a constant temperature, eliminating the need for thermocycling equipment. It is typically more robust, specific, and sensitive than PCR. This literature review summarizes LAMP primer design, reaction protocol development, sensitivity and specificity testing, and result detection methods. We provide examples of how LAMP's advantages are exploited in disease diagnosis and survey its diverse applications in plant pathogenic fungi research. These applications include the detection, identification, and monitoring of plant pathogenic fungi; the replacement of culture-based methods; the detection of genetic regions associated with functional changes; and the detection of single nucleotide polymorphisms. A comprehensive list of available assays is also provided. Despite its shortcomings-including difficulties with primer design, risks of cross-contamination, and the potential for false positives-LAMP holds significant potential to gain widespread recognition and popularity in the study of plant pathogenic fungi.

Keywords: DNA-based detection; SNP detection; assay development; identification; in-field diagnostics; pathogen detection.

Figure 1

(1) Target DNA and a strand displacement polymerase (shown as a light blue shape) are present in the reaction mix. (2) Firstly, the forward inner primer (FIP) binds to the target DNA with its complementary 3’ end, while DNA polymerase displaces the opposite strand on the original target and polymerization begins and (3) continues in 3’–5’ direction. DNA ends elongated on their 3’ ends are marked as arrows. (4) Then, F3 primer binding and (5) polymerization are also initiated from F3. Due to the activity of the polymerase, the DNA generated in the previous step is displaced. (6) This partially releases the previously synthesized fragment containing FIP. As FIP contains a region complementary to the same DNA strand, the DNA will be self-joined through base pairing to form a loop. (7–10) The same reaction steps also take place on the opposite target strand, involving backward inner primer (BIP) and B3. (11) A DNA fragment with loops on both ends is formed. This structure provides the basis for the upcoming DNA amplification steps. During the amplification, (12) FIP binds to the original target DNA and (13–14) its free 3’ ends, and those of the previously synthesized strands are also elongated. (15–16) Similarly, elongation takes place from the 3’ ends of the later synthesized strands after loop formation. (17–20) In the next steps, LAMP results in the formation of several DNA fragments with different structures, with varying amounts of loops and different DNA lengths. Labels are omitted from steps 17–20 for simplicity.