**2.1 Amplification – LAMP**

Since the publication of the first report regarding LAMP in 2000 (Notomi et al., 2000), LAMP has been used to detect different kinds of pathogens (Mori & Notomi, 2009), including viruses (Kubo et al., 2010), bacteria (Iwamoto et al., 2003), and protozoa (Spencer et al., 2010), and thus far, approximately 500 reports have been published regarding the application of LAMP. Because the LAMP method is simple and quick, it has been considered one of the most ideal nucleic acid amplification methods, which can be applied as an easy-to-use and cost-effective genetic test system (Parida et al., 2008).

#### **2.1.1 Mechanism of LAMP**

Although the reaction mechanism appears complicated, LAMP is simple to perform—it involves mixing primers (designed as depicted in figure 1-A), DNA polymerase with stranddisplacement activity, and dNTPs, in a buffer containing magnesium ions, and maintaining the mixture at a constant temperature of 60–67 °C for 15–60 minutes. If template DNA molecules are present in the sample solution, large quantities of DNA with the target sequence (amplicon) are produced after incubation.

Figures 1-B and C show the schematic representation of the mechanism of LAMP. First, the forward inner primer (FIP) anneals to the template DNA at the F2c sequence and the extension reaction occurs by the enzymatic activity of *Bst* polymerase. Because *Bst* polymerase exhibits strand displacement activity, the product obtained from FIP is displaced by the other extension reaction associated with the F3 primer. Subsequently, the extension reaction occurs from the backward inner primer (BIP) on the product of the FIP, and not on the template DNA with a B2c sequence; the product obtained is also displaced by DNA synthesis associated with the B3 primer. These reactions result in a product with a dumbbell-like structure as shown in figure 1-B. The formation of the dumbbell-like product is essential for LAMP to establish isothermal amplification because the loop structures are always single stranded and can be annealed by FIP or BIP. Thus, formation of the loop structure can lead to the elimination of the denaturing step, which is otherwise essential in PCR for obtaining single-stranded DNA.

After the formation of the dumbbell-like structure, a cyclic reaction is spontaneously established between the dumbbell-like structure and its complementary product, as shown in figure 1-C. Furthermore, in the course of the cyclic reaction, elongated products with various copies of the target sequence are also produced.

The basic characteristics of the LAMP method are summarized below:


Since the publication of the first report regarding LAMP in 2000 (Notomi et al., 2000), LAMP has been used to detect different kinds of pathogens (Mori & Notomi, 2009), including viruses (Kubo et al., 2010), bacteria (Iwamoto et al., 2003), and protozoa (Spencer et al., 2010), and thus far, approximately 500 reports have been published regarding the application of LAMP. Because the LAMP method is simple and quick, it has been considered one of the most ideal nucleic acid amplification methods, which can be applied

Although the reaction mechanism appears complicated, LAMP is simple to perform—it involves mixing primers (designed as depicted in figure 1-A), DNA polymerase with stranddisplacement activity, and dNTPs, in a buffer containing magnesium ions, and maintaining the mixture at a constant temperature of 60–67 °C for 15–60 minutes. If template DNA molecules are present in the sample solution, large quantities of DNA with the target

Figures 1-B and C show the schematic representation of the mechanism of LAMP. First, the forward inner primer (FIP) anneals to the template DNA at the F2c sequence and the extension reaction occurs by the enzymatic activity of *Bst* polymerase. Because *Bst* polymerase exhibits strand displacement activity, the product obtained from FIP is displaced by the other extension reaction associated with the F3 primer. Subsequently, the extension reaction occurs from the backward inner primer (BIP) on the product of the FIP, and not on the template DNA with a B2c sequence; the product obtained is also displaced by DNA synthesis associated with the B3 primer. These reactions result in a product with a dumbbell-like structure as shown in figure 1-B. The formation of the dumbbell-like product is essential for LAMP to establish isothermal amplification because the loop structures are always single stranded and can be annealed by FIP or BIP. Thus, formation of the loop structure can lead to the elimination of the denaturing step, which is otherwise essential in

After the formation of the dumbbell-like structure, a cyclic reaction is spontaneously established between the dumbbell-like structure and its complementary product, as shown in figure 1-C. Furthermore, in the course of the cyclic reaction, elongated products with

1. The whole amplification reaction occurs continuously under isothermal conditions, thus eliminating the need to use a thermal cycler, which is commonly used for PCR. 2. Because LAMP primers recognize 6 distinct regions, the specificity of LAMP is much

3. Amplification can be performed using an RNA template only by the addition of reverse

4. The LAMP reaction can be accelerated by using additional primers, called "loop primers," which are designed between F1c/B1c and F2c/B2c (Nagamine et al.,

as an easy-to-use and cost-effective genetic test system (Parida et al., 2008).

**2. Steps involved in molecular diagnostics** 

sequence (amplicon) are produced after incubation.

PCR for obtaining single-stranded DNA.

2002).

various copies of the target sequence are also produced.

transcriptase to the reaction (one-step RT-LAMP).

The basic characteristics of the LAMP method are summarized below:

higher than that of the other commonly used amplification techniques.

**2.1 Amplification – LAMP** 

**2.1.1 Mechanism of LAMP** 

Novel Molecular Diagnostic Platform for Tropical Infectious Diseases 449

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

The sample processing method is the next important step in molecular diagnostics. Silicabased methods are well known and have been applied to a wide variety of samples, including blood and tissue (Bendall, 2002). However, these methods are unsuitable for resource-limited facilities due to the cumbersome procedures involved, including washing with organic solvents using high-speed centrifugation. Therefore, we have developed a simple and swift sample processing method named PURE. Thus far, it has been confirmed that PURE can be successfully applied to sputum, blood, serum, and swab samples. The

1. An aliquot of sample (blood, sputum, etc.) is added to the alkaline-based extraction

2. The sample solution is treated with adsorbent powder to remove inhibitory materials contained in samples and to neutralize the solution without any loss of target DNA. 3. After separating the solution from the powder by filtration, the obtained filtrate containing target DNA molecules can be used for reconstituting dried LAMP reagents,

**2.2 Detection – Calcein method** 

et al., 2009; Mori et al., 2006).

Fig. 2. Mechanism involved using calcein

mechanism of PURE is described below:

solution and treated by heat to lyse the pathogens.

which are deposited to the lids of LAMP reaction tubes.

**2.3 Sample preparation – PURE** 

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

A) Design of the LAMP primers

B) Formation of a dumbbell-like structure

C) Cyclic and elongation reactions
