Colorimetric detection of loop-mediated isothermal amplification

Short Technical Reports


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Colorimetric detection of loopmediated isothermal amplification reaction by using hydroxy naphthol blue Motoki Goto1, Eiichi Honda2, Atsuo Ogura1, Akio Nomoto3, and Ken-Ichi Hanaki1 1Section of Animal Research, Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan, 2Department of Veterinary Medicine, Tokyo University of Agriculture and Technology, Tokyo, Japan, and 3Department of Microbiology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.

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BioTechniques 46:167-172 (March 2009) doi 10.2144/000113072 Keywords: colorimetric assay; hydroxy naphthol blue; HNB; loop-mediated isothermal amplification; LAMP

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 Vol. 46 | No. 3 | 2009

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 (2–6); (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

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Figure 1. Effect of Mg2+ ion concentration on the spectra of HNB in the LAMP reaction solution. (A) Absorption spectra of HNB in the LAMP reaction solutions with various Mg2+ ion concentrations. Mg2+ ion concentrations: red line, 8 mM; cyan line, 7 mM; magenta line, 6 mM; blue line, 5 mM; green line, 4 mM; and black line, 0 mM. A photograph in the graph shows the true color of HNB in the reaction solution. Tube a, 8 mM Mg2+ ions without dNTPs; tube b, 8 mM Mg2+ ions with dNTPs; tube c, 7 mM Mg2+ ions; tube d, 6 mM Mg2+ ions; tube e, 5 mM Mg2+ ions; tube f, 4 mM Mg2+ ions; and tube g, 0 mM Mg2+ ions. (B) Correlation between the absorbance of HNB and Mg2+ ion concentration. Black line, 600 nm; green line, 610 nm; blue line, 620 nm; magenta line, 630 nm; cyan line, 640 nm; crimson line, 650 nm; red line, 660 nm; gray line, 670 nm; yellow line, 680 nm.

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 (2–4). Several studies have reported the use of the LAMP method for detecting various pathogens (2–6,10–15). However, many of these studies used an expensive real-time turbidimeter (2,4–6,10,12) 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 costwww.BioTechniques.com


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 Mg 2+ 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 Mg 2+ 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/ Vol. 46 | No. 3 | 2009

μM each of F3 and B3, 0.8 μM each of LF and LB, 1.4 mM of each dNTP, 120 μM HNB, and MgSO4 at various concentrations in the LAMP buffer [10 mM KCl, 10 mM (NH4)2SO4, 20 mM Tris-HCl (pH 8.8 at 25°C), 0.1% Tween 20, and 0.8 M betaine]. An absorption spectra analysis was performed in a 0.2-mL quartz cuvette with a 10-mm path length by using the spectrophotometer V530 with Spectra Manager software v1.54 (Jasco, Hachioji, Tokyo, Japan). The instrument was first set to zero at 750 nm for distilled water, and absorbance in the range of 750 nm to 350 nm was recorded at 1-nm intervals.

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Figure 2. Comparative sensitivity of LAMP assay using different dyes for the detection of serially diluted λ DNA. (A) Visualization on a light box. (a) HNB: The color changes from violet (negative reaction) to sky blue (positive reaction). (b) SYBR green and (c) calcein: The color changes from orange (negative reaction) to yellow (positive reaction). (B) Visualization under UV irradiation. (b) SYBR green and (c) calcein: Bright fluorescence indicates a positive reaction. Tube 1, 1:103 dilution (1.58 × 107 copies/tube); tube 2, 1:104 dilution; tube 3, 1:105 dilution; tube 4, 1:106 dilution; tube 5, 1:107 dilution; tube 6, 1:108 dilution; tube 7, 1:109 dilution; and tube 8, no template.

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′-CAGCATCCCTTTCGG C ATACC AG G TG G C A AG G G TAATGAGG-3′; backward inner primer (BIP), 5′-GGAGGTTGAAGAACTGCG G C AG TCG ATG G CG T TCG TACTC-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). Spectrophotometric analysis The LAMP reaction solution used for spectrophotometric analysis consisted of the following components: 0.88 ng λ DNA, 1.6 μM each of FIP and BIP, 0.2

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LAMP assays in 0.2-mL PCR tubes For comparing the detection sensitivity among different colorimetric LAMP assays, three different reaction mixtures were prepared. A LAMP assay with HNB was carried out in a 25-μL reaction mixture containing 2 μL of 10-fold serially diluted λ DNA, 1.6 μM each of FIP and BIP, 0.2 μM each of F3 and B3, 0.8 μM each of LF and LB, 1.4 mM of each dNTP, 8 U of the large fragment of Bst DNA polymerase (New England BioLabs, Sumida, Tokyo, Japan), 8 mM MgSO4, and 120 μM HNB in the LAMP buffer. A second LAMP assay with SYBR green was carried out in a 25-μL reaction mixture containing the same components described, except excluding the HNB. A third LAMP assay with calcein was carried out in a 25-μL reaction mixture, in which HNB was replaced with 25 μM calcein and 0.5 mM MnCl 2 , as described in a previous report (18). Reaction mixtures without λ DNA were also prepared to serve as negative controls. All reaction mixtures were incubated at 63°C for 1 h and then heated at 80°C for 2 min to terminate the reaction in a Mastercycler ep gradient S thermocycler (Eppendorf, Hamburg, Germany). For the visualization of the LAMP reaction with SYBR green, 10 μL of SYBR green I (Invitrogen) diluted 1:100 was added to each tube. The reaction tubes were placed on a light box, and photographs were obtained using a digital camera (C-750 UZ; Olympus, Shinjuku, Tokyo, Japan). The reaction tubes were also irradiated at 365 nm with a handheld UV lamp (UVGL-58; UVP, Upland, CA, USA), and photographs were obtained. LAMP assay in a 96-well plate The LAMP reaction mixtures containing HNB and serially diluted λ DNA were prepared as described in “LAMP assays in 0.2-mL PCR tubes.” Each 30-μL reaction mixture was added to the wells of a 96-well www.BioTechniques.com


microplate (Catalog no. 353912; BD, Minato, Tokyo, Japan) in quintuplicate. The plate was sealed with a ThermalSeal RT sealing film (Excel Scientific, Victorville, CA, USA) and then overlaid with a polyethylene gel sheet with a thickness of 3 mm (Catalog no. 01001063138; Tokyu Hands, Shibuya, Tokyo, Japan) that was prewarmed at 63°C in an incubator (HB-100; Taitec, Koshigaya, Saitama, Japan). The plate with the polyethylene gel sheet was incubated at 63°C for 1 h in the incubator and then cooled down at room temperature for 5 min. After removing the gel sheet, the absorbance of each well was read at 650 nm with a microplate reader (ARVO MX-fa; PerkinElmer, Yokohama, Kanagawa, Japan). The plate was then placed on a light box, and a photograph was taken.

Results and discussion

HNB was reported to be useful as a colorimetric indicator for the titration of Ca2+ ions at pH 13.0 and Mg2+ ions at pH 10.0 (19). Therefore, we hypothesized that HNB could be a novel indicator for the LAMP reaction by monitoring the change in the Mg 2+ ion concentration since the large fragment of Bst DNA polymerase synthesizes DNA under alkaline conditions (pH 8.8 at 25°C). The color of HNB changes depending on the pH of the solution; when the solution contained 8 mM Mg2+ ions and no dNTPs, its color was magenta (Figure 1A, tube a) at pH 8.6–9.0 and violet at pH 8.4 (data not shown). After the addition of 1.4 mM dNTPs to this solution, the color of HNB changed from magenta to violet (Figure 1A, tube b) irrespective of the pH. This color change is induced by the chelation of Mg2+ ions by dNTPs (20). We measured the absorbance spectra of the LAMP reaction solutions containing various concentrations of Mg 2+ ions when HNB was used as the colorimetric indicator. In the absence of Mg 2+ ions, solutions containing HNB exhibited an absorbance peak at 650 nm, and the absorbance values decreased as the Mg2+ ion concentration increased (Figure 1A, graph). The absorbance of solutions containing both HNB and Mg 2+ ions was also measured at different absorbance wavelengths ranging from 600 to 680 nm at 10-nm intervals (Figure 1B). The absorbance of the solutions was correlated with the Mg 2+ ion concentration, and the correlation showed that an absorbance wavelength from 630 to 670 nm, especially 650 nm, was suitable for LAMP assay using a microplate reader. Vol. 46 | No. 3 | 2009

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Figure 3. LAMP method adapted to a 96-well plate format. (A) Color change in a 96-well plate. Column 1, 1:103 dilution (1.58 × 107 copies/well); column 2, 1:104 dilution; column 3, 1:105 dilution; column 4, 1:106 dilution; column 5, 1:107 dilution; column 6, 1:108 dilution; column 7, 1:109 dilution; and column 8, no template (negative control). Violet-colored and sky blue–colored wells showed negative and positive reactions, respectively. (B) A graph showing the absorbance of the LAMP reaction solution containing HNB at 650 nm. The numbers correspond to those in Figure 3A. Yellow columns show the mean absorbance values of LAMP reactions (n = 5). Red circles indicate the absorbance of individual LAMP reaction solutions. The dashed horizontal line indicates the cut-off value that was calculated by adding two standard deviations to the mean of the negative control.

The performances of LAMP assays using HNB, SYBR green, and calcein were compared with regard to the detection of 10-fold serially diluted λ DNA. The color change of HNB from violet to sky blue indicated a positive reaction, and the LAMP assay using HNB could detect λ DNA at dilutions ≤1:108 (160 copies/ tube) (Figure 2A, tube a). The detection sensitivity was equivalent to that of the assay using SYBR green (Figure 2A, tube b). This result implied that HNB at 120 μM did not interfere with DNA synthesis by the large fragment of Bst DNA polymerase. In contrast, the detection sensitivity of the LAMP assay using calcein was 10 times lower than those of other assays under both daylight (Figure 2A, tube c) and UV light (Figure 2B, tube c). The brightness of calcein fluorescence was significantly weaker than that of SYBR green fluorescence (Figure 2B). For the colorimetric assay using

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calcein, Mn 2+ ions should be added to the pre-reaction solution (18). However, we confirmed that the LAMP assay using SYBR green was inhibited with addition of 0.5 mM Mn2+ ions to the pre-reaction solution, and the detection sensitivity was 10 times lower than that of the original (data not shown). The LAMP assay using HNB was superior to the other assays because (i) opening the reaction tube was not required to determine whether the reaction was positive or negative (this reduces the risk of cross-contamination); (ii) detection sensitivity was equivalent to that of the assay using SYBR green; and (iii) the positive/negative result of the LAMP reaction could be easily judged by the naked eye. Each assay was carried out in quintuplicate, and the results were well reproduced. Absorbance of the solution containing HNB at 650 nm adequately reflected the Mg 2+ ion concentration in the solution (Figure 1B); therefore, a 96-well plate format assay using a microplate reader with a 650-nm filter was developed to make this LAMP assay a high-throughput technique. First, we examined the type of 96-well plates that would be suitable for the LAMP reaction. Polystyrene plates were unsuitable for the reaction because heat conduction was not uniform among the wells. An unexpected color change was observed in the negative controls containing HNB when TopYield strips (Thermo Fisher Scientific, Bunkyo, Tokyo, Japan) composed of polycarbonate and designed for immuno-PCR were used for the assay. The cause of this color change could not be determined. Therefore, the 96-well thin-wall plate composed of polypropylene was selected. However, condensation occurring on the inside of the top cover of the plate hindered the correct measurement of absorbance values. The condensation was prevented by simply overlaying a prewarmed (to 63°C) polyethylene gel sheet on the top cover. The LAMP assay using HNB was performed in the 96-well thin-wall plate in an incubator at 63°C for 1 h. The sensitivity as judged by the naked eye (Figure 3A) was equivalent to that observed when using 0.2-mL PCR tubes (1:108 dilution) incubated in a thermal cycler at 63°C for 1 h (Figure 2A, tube a). The absorbance of each well was also measured at 650 nm by using a microplate reader (Figure 3B). The reactions could not be quantified by the absorbance measurement, even though positive/negative reactions could be identified qualitatively. Higher absorbance values in the positive reactions were caused by the precipitation of the www.BioTechniques.com


byproduct magnesium pyrophosphate in the optical path. The colors of HNB in the solutions with positive/negative reactions were stable after ≥2 weeks of exposure to ambient light. The new colorimetric assay for LAMP can be carried out by only adding HNB to the reaction solution at the final concentration of 120 μM. This colorimetric LAMP assay using HNB in both tubes and 96-well plates has some benefits compared with other nucleic acid amplification techniques: easy operation, no need for special equipment, superior sensitivity and speed, low contamination risk, and suitability for high-throughput DNA and RNA detection. Therefore, this colorimetric assay is suitable not only for laboratory research but also for clinical diagnoses of many infectious diseases.

Acknowledgements

This study was supported by the Ministry of Health, Labour, and Welfare of Japan (grant no. H20-Emerging-General-006). The authors declare no competing interests.

References

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