Loop Mediated Isothermal Amplification: Principles and Applications in Plant Virology

Abstract

In the last decades, the evolution of molecular diagnosis methods has generated different advanced tools, like loop-mediated isothermal amplification (LAMP). Currently, it is a well-established technique, applied in different fields, such as the medicine, agriculture, and food industries, owing to its simplicity, specificity, rapidity, and low-cost efforts. LAMP is a nucleic acid amplification under isothermal conditions, which is highly compatible with point-of-care (POC) analysis and has the potential to improve the diagnosis in plant protection. The great advantages of LAMP have led to several upgrades in order to implement the technique. In this review, the authors provide an overview reporting in detail the different LAMP steps, focusing on designing and main characteristics of the primer set, different methods of result visualization, evolution and different application fields, reporting in detail LAMP application in plant virology, and the main advantages of the use of this technique.

Keywords: Bst DNA polymerase; LAMP; loop-mediated isothermal amplification; plant virology; primers; real-time monitoring; viroids; virus.

Figure 1

Typical map for loop mediated isothermal amplification (LAMP) primer set positioning and corresponding sequence homology. For each primer, the same color represents the corresponding sequence in the target. The primer sequence corresponds to the reverse complementary sequence of the target region. (FIP: forward inner primer; F3: forward outer primer; FL: loop primer F-Loop; BL: loop primer B-Loop; B3: backward outer primer; BIP: backward inner primer).

Figure 2

Typical electrophoretic analysis on a 2% agarose gel followed by ethidium bromide (EtBr) staining of LAMP amplified products, using a specific primer set. The LAMP method forms amplified products of various sizes (“ladder pattern”), consisting of alternately inverted repeats of the target sequence on the same strand. Lane M: 1 kb ladder (Nippon Genetics); lanes 2–6: positive LAMP reaction samples; lane C+: positive control; lane C−: negative control.

Figure 3

Different naked-eye results inspection. (A) Naked-eye observation under natural light of the reaction tube after addition of ethidium bromide in the reaction mixture for positive result discrimination. In the case of a negative result, a wine coloration is shown (left), while in a positive sample, the reaction mixture shows a salmon pink coloration (right). (B) Naked-eye observation under natural light of the reaction tube with Picogreen fluorescent dye in LAMP assay amplification. In the case of a negative result, the original orange color is retained (left), whereas in the case of a positive amplification, the original color of the dye changes permanently to yellow (right). (C) Result discrimination by ion indicator: (left) negative control that did not contain template DNA, showing clear mixture; (right) increases in turbidity observed on positive sample. (D) Detection of target DNA in LAMP reaction under UV light, using calcein as fluorescent metal indicator. (Left) negative control; (right) emitted fluorescence by positive sample owing to the interaction of calcein with residual magnesium ion. (E) Result discrimination by HNB (hydroxy naphthol blue): the color change of the mixture is from violet on the negative reaction (left) to sky blue on the positive reaction (right).

Figure 4

Detection using calcein as a fluorescent metal indicator. In the DNA amplification process, pyrophosphate ions are produced as a by-product from the reaction substrate deoxyribonucleotide triphosphates (dNTPs). In the presence of target DNA, during the LAMP reaction, newly generated pyrophosphate ion (P₂O₇⁴⁻) deprives calcein of manganous ion, which combines with residual magnesium ion (Mg²⁺), producing greater fluorescence.

Figure 5

Result comparison between real-time LAMP and conventional real-time polymerase chain reaction (PCR). Panel (A) shows the LAMP amplification curve, which looks like a “hat”, while panel (B) shows the conventional sigmoidal real-time PCR amplification curve.

Figure 6

LAMP combined with lateral flow assay (LFA): the LAMP reaction is performed using a biotinylated forward inner primer (FIP) primer. After 30–60 min of initial incubation at constant temperature (60–65° C), a specific probe labelled with FITC (fluorescein isothiocyanate) is added to the reaction mixture and incubated for 10 min at the same temperature; in this step, dual-labeled LAMP product is produced. Subsequently, the reaction mixture is mixed with a detection buffer containing rabbit anti-FITC antibodies coupled with colloidal gold, and the LFD (Lateral-Flow Dipstick) strip is inserted into the tube. In a positive sample, the LAMP product complex moves through the LFA pad (an absorbent pad or strip) and binds with anti-FITC antibodies. The results can be read in a few minutes, just visualizing the pad. In the case of a negative reaction, no products are generated. An anti-rabbit antibody at the test control band retains some of the unbound gold conjugated antibody and produces a band that should always be visible.