Norihiro Tomita

Loop-mediated isothermal amplification (LAMP) of gene sequences and simple visual detection of products

As the human genome is decoded and its involvement in diseases is being revealed through postgenome research, increased adoption of genetic testing is expected. Critical to such testing methods is the ease of implementation and comprehensible presentation of amplification results. Loop-mediated isothermal amplification (LAMP) is a simple, rapid, specific and cost-effective nucleic acid amplification method when compared to PCR, nucleic acid sequence-based amplification, self-sustained sequence replication and strand displacement amplification. This protocol details an improved simple visual detection system for the results of the LAMP reaction. In LAMP, a large amount of DNA is synthesized, yielding a large pyrophosphate ion by-product. Pyrophosphate ion combines with divalent metallic ion to form an insoluble salt. Adding manganous ion and calcein, a fluorescent metal indicator, to the reaction solution allows a visualization of substantial alteration of the fluorescence during the one-step amplification reaction, which takes 30–60 min. As the signal recognition is highly sensitive, this system enables visual discrimination of results without costly specialized equipment. This detection method should be helpful in basic research on medicine and pharmacy, environmental hygiene, point-of-care testing and more.

Loop-mediated isothermal amplification (LAMP): principle, features, and future prospects

Loop-mediated isothermal amplification (LAMP), a newly developed gene amplification method, combines rapidity, simplicity, and high specificity. Several tests have been developed based on this method, and simplicity is maintained throughout all steps, from extraction of nucleic acids to detection of amplification. In the LAMP reaction, samples are amplified at a fixed temperature through a repetition of two types of elongation reactions occurring at the loop regions: self-elongation of templates from the stem loop structure formed at the 3′-terminal and the binding and elongation of new primers to the loop region. The LAMP reaction has a wide range of possible applications, including point-of-care testing, genetic testing in resource-poor settings (such as in developing countries), and rapid testing of food products and environmental samples.

Clinical Evaluation of a Loop-Mediated Amplification Kit for Diagnosis of Imported Malaria

Background. Diagnosis of malaria relies on parasite detection by microscopy or antigen detection; both fail to detect low-density infections. New tests providing rapid, sensitive diagnosis with minimal need for training would enhance both malaria diagnosis and malaria control activities. We determined the diagnostic accuracy of a new loop-mediated amplification (LAMP) kit in febrile returned travelers. Methods. The kit was evaluated in sequential blood samples from returned travelers sent for pathogen testing to a specialist parasitology laboratory. Microscopy was performed, and then malaria LAMP was performed using Plasmodium genus and Plasmodium falciparum–specific tests in parallel. Nested polymerase chain reaction (PCR) was performed on all samples as the reference standard. Primary outcome measures for diagnostic accuracy were sensitivity and specificity of LAMP results, compared with those of nested PCR. Results. A total of 705 samples were tested in the primary analysis. Sensitivity and specificity were 98.4% and 98.1%, respectively, for the LAMP P. falciparum primers and 97.0% and 99.2%, respectively, for the Plasmodium genus primers. Post hoc repeat PCR analysis of all 15 tests with discrepant results resolved 4 results in favor of LAMP, suggesting that the primary analysis had underestimated diagnostic accuracy. Conclusions. Malaria LAMP had a diagnostic accuracy similar to that of nested PCR, with a greatly reduced time to result, and was superior to expert microscopy.

Novel Molecular Diagnostic Platform for Tropical Infectious Diseases

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 chapter, we would like to describe a recently developed novel diagnostic platform and discuss its application for realizing molecular diagnostics for infectious diseases within resourcelimited 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.