**2.2 Detection – Calcein method**

448 Current Topics in Tropical Medicine

Fig. 1. Schematic representation of the mechanism of the LAMP assay

Conventional LAMP reagents are supplied in liquid form, and they have to be stored below -20 °C, similar to most PCR reagents. However, because of the lack of a freezer and cold chain transportation system in most of the peripheral laboratories in developing countries, it is essential to formulate LAMP reagents, which can be stably preserved at ambient temperatures (Jorgensen et al., 2006; Aziah et al., 2007). The newly formulated LAMP reagents are dried down into the lid of the reaction tubes, thus obtaining preservation stability at ambient temperatures for more than 12 months. The dried LAMP reagents can be reconstituted quite easily by shaking the tubes after the addition of the purified DNA solution. Because the LAMP reagent for each reaction is deposited on the individual tubes in advance, there is no longer a need to prepare and dispense master-mix solutions to the reaction tubes. Thus, liquid handling using micropipette, one of the most skillful steps, becomes unnecessary in the course of the assay. Moreover, this can contribute to reduced

**2.1.2 Strategies that make LAMP simple and cost-effective**

A) Design of the LAMP primers

B) Formation of a dumbbell-like structure C) Cyclic and elongation reactions

risk of carryover contamination during the assay.

The results of the LAMP assay can be detected visually by observing the strength of the green fluorescence emitted after the reaction. Figure 2 represents the mechanism of the calcein method (Tomita et al., 2008). Before LAMP amplification, the metalochrome indicator "calcein" is quenched by the effect of a manganese ion. After the LAMP reaction, pyrophosphate ions (PPi) are produced as a by-product of polymerase reaction; PPi subsequently forms a manganese pyrophosphate complex, causing the removal of the manganese ion from calcein, because the PPi are a stronger base than calcein. Next, free calcein combines with a magnesium ion to produce bright fluorescence. This technology enables the detection of LAMP reactions without the use of fluorescence detectors, which are usually expensive and difficult to manage in resource-limited settings. Other technologies for visual detection using LAMP have also been reported (Tao et al., 2011; Goto et al., 2009; Mori et al., 2006).

Fig. 2. Mechanism involved using calcein