Introduction
Infectious disease is one of the most concerning health issues worldwide. To provide patients with effective medical treatment and prevent the spread of diseases and emergence of drug-resistant strains, quick and reliable diagnostic techniques are in high demand. However, lack of accessibility to such diagnostic systems has resulted in the deterioration of the situation in most developing countries, especially in sub-Saharan tropical countries (Rodrigues et al., 2010). Diagnostics using molecular technologies have emerged as a promising methodology because of their remarkable high sensitivity, and therefore, they have been applied as diagnostic tools for detecting various kinds of pathogens in clinical settings in developed countries. However, resources essential for molecular assays, such as bio-safety cabinets, a stable supply of electricity, and well-experienced technicians, are scarce in most of the peripheral laboratories in developing countries. In this topic, we would like to describe a recently developed novel diagnostic platform and discuss its application for realizing molecular diagnostics for infectious diseases within resource-limited settings.
Molecular diagnostics comprise the following 3 steps: sample preparation, amplification, and detection. To develop a molecular diagnostic platform with the desired simplicity and performance, it is necessary to introduce element technologies for all the 3 steps, which are less complicated and can be used in peripheral laboratories with limited resources. Of the abovementioned 3 steps, amplification of target DNA/RNA is the most important. Therefore, the loop-mediated isothermal amplification (LAMP) method involving the calcein detection method has been applied to the platform as a key technology. LAMP, using the calcein method, enables recognition of small quantities of DNA/RNA of pathogens present in clinical specimens by means of the fluorescence emitted from the LAMP solutions after amplification.
The next important step is sample processing, for which we have developed a simple and easy-to-use technology, namely, procedure for ultra rapid extraction (PURE). The combination of both these technologies can be considered a novel platform for molecular diagnostics, which can be applied to resource-limited settings. The fundamental characteristics of these element technologies and application of the novel platform to diagnostics for evaluation of certain tropical diseases are discussed below.
Steps involved in molecular diagnostics
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).
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 strand-displacement 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:
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 higher than that of the other commonly used amplification techniques.
3. Amplification can be performed using an RNA template only by the addition of reverse transcriptase to the reaction (one-step RT-LAMP).
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., 2002).
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
