Loop-mediated isothermal amplification (LAMP), a novel gene amplification method, enables the synthesis of larger amounts of both DNA and a visible byproduct—namely, magnesium pyrophosphate—without thermal cycling. A positive reaction is indicated by the turbidity of the reaction solution or the color change after adding an intercalating dye to the reaction solution, but the use of such dyes has certain limitations. Hydroxy naphthol blue (HNB), a metal indicator for calcium and a colorimetric reagent for alkaline earth metal ions, was used for a new colorimetric assay of the LAMP reaction. Preaddition of 120 µM HNB to the LAMP reaction solution did not inhibit amplification efficiency. A positive reaction is indicated by a color change from violet to sky blue. The LAMP reaction with HNB could also be carried out in a 96-well microplate, and the reaction could be measured at 650 nm with a microplate reader. The colorimetric LAMP method using HNB would be helpful for high-throughput DNA and RNA detection.

Introduction

Loop-mediated isothermal amplification (LAMP), a novel gene amplification method, is an autocycling and strand displacement DNA synthesis method involving the use of the large fragment of Bst DNA polymerase and a set of four specially designed primers (1). Gene amplification by the LAMP method is superior to that by PCR for the following reasons: (i) LAMP does not require an expensive thermocycler because all reactions can be performed at a constant temperature ranging from 60°C to 65°C; (ii) the amplification specificity is extremely high because the LAMP reaction requires a set of four oligonucleotide primers that recognize six distinct regions on the target DNA; (iii) the detection limit of LAMP is expected to be equal to or higher than that of PCR, and the detection time is shorter (2,3,4,5,6); (iv) the LAMP reaction can be accelerated by using two specially designed loop primers (7); and (v) visualization of DNA products on gel electrophoresis is not required for assessing successful DNA amplification because a positive LAMP reaction causes the solution to become cloudy due to the formation of the magnesium pyrophosphate byproduct (8). The turbidity of the solution has a high correlation with the amount of DNA synthesized, and a real-time turbidimeter for the LAMP reaction was developed for quantifying initial template DNA (9). LAMP can also be adapted to RNA amplification by simply adding a reverse transcriptase in the reaction solution (2,3,4).

Several studies have reported the use of the LAMP method for detecting various pathogens (2,3,4,5,6,10,11,12,13,14,15). However, many of these studies used an expensive real-time turbidimeter (2,4,5,6,10,12) or a real-time PCR system (3,11,13,15) for the reaction confirmation. The use of expensive equipment decreases the versatility of LAMP and greatly limits the wide use of this procedure, especially in developing countries. Detection of turbidity by the naked eye is the simplest and most cost-efficient method for judging a positive or negative LAMP reaction, although this method requires some skill for assessing the result. For better visibility of the reaction result, a DNA intercalating dye such as SYBR green (16,17), Picogreen (3,14), or propidium iodide (17) is added to the solution after the reaction is completed. When the LAMP reaction is positive, a color change is observed under ambient light. However, as in the case of analysis in gel electrophoresis, the colorimetric assay using the intercalating dye is associated with an increased risk of contamination of other subsequent LAMP reaction solutions because the assay requires opening of the tubes. To avoid such contamination, separate rooms should be used for LAMP setup and analysis.

The LAMP reaction results in large amounts of pyrophosphate ion byproduct; these ions react with Mg²⁺ ions to form the insoluble product magnesium pyrophosphate. Since Mg²⁺ ion concentration decreases as the LAMP reaction progresses, the LAMP reaction can be quantified by measuring the Mg²⁺ ion concentration in the reaction solution. On the basis of this phenomenon, Tomita et al. (18) developed a simple colorimetric assay for the detection of the LAMP reaction by adding calcein, a fluorescence metal indicator, to the pre-reaction solution. Here, we report a simpler colorimetric assay for the detection of the LAMP reaction by using another metal ion indicator, namely, hydroxy naphthol blue (HNB). This colorimetric assay is superior to the existing colorimetric assays for LAMP with regard to reducing contamination risks, and is helpful in high-throughput DNA and RNA detection.

Materials and methods

Materials

HNB (CAS No. 63451–35–4) and calcein (CAS No. 1461–15–0) were purchased from Dojindo (Kamimashiki, Kumamoto, Japan). HNB was dissolved in distilled water at 20 mM to prepare a stock solution. Calcein was first dissolved in dimethyl sulfoxide at 5 mM, and then a stock solution consisting of 0.5 mM calcein and 10 mM MnCl₂ was prepared with distilled water. The lambda DNA (λ DNA) used as the template in this study was purchased from Nippon Gene (Toyama, Japan). A 10-fold serially diluted λ DNA solution was prepared with distilled water from the stock solution (0.44 µg/µL; 7.92 × 10⁹ copies/µL). Six oligonucleotide primers were designed using the PrimerExplorer V4 software available on the web site (http://primerexplorer.jp/e): forward inner primer (FIP), 5′-CAGCATCCCTTTCGGCATACCAGGTGGCAAGGGTAATGAGG-3′; backward inner primer (BIP), 5′-GGAGGTTGAAGAACTGCGGCAGTCGATGGCGTTCGTACTC-3′; forward outer primer (F3), 5′-GAATGCCCGTTCTGCGAG-3′; backward outer primer (B3), 5′-TTCAGTTCCTGTGCGTCG-3′; loop forward primer (LF), 5′-GGCGGCAGAGTCATAAAGCA-3′; and loop backward primer (LB), 5′-GGCAGATCTCCAGCCAGGAACTA-3′. These primers were synthesized by Invitrogen (Minato, Tokyo, Japan).