Loop-mediated Isothermal Amplification Reaction Using a Nondenatured Template

Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method that amplifies DNA with high specificity, efficiency, and rapidity under isothermal conditions (1). The LAMP method requires a set of four specially designed primers and a DNA polymerase with strand displacement activity. The amplification products are stem-loop DNA structures with several inverted repeats of the target and cauliflower-like structures with multiple loops, yielding >500 μg/mL. Although LAMP amplifies DNA under isothermal conditions, the template DNA is heat-denatured. To determine whether LAMP can be performed under isothermal conditions at all steps, we attempted to amplify DNA without using a heat-denatured template. Hepatitis B virus (HBV) DNA that was obtained from a patient was digested with BamHI and cloned into pBR322 plasmid vector. Plasmid and genomic DNA was prepared using the plasmid Midi reagent set (Qiagen) and EXTRAGEN reagent set (Tosoh), respectively. LAMP amplification of HBV DNA was performed with a pair of primers as described previously (1). The DNA was amplified in a 25-μL reaction mixture containing 1.6 μM each of FIP and BIP, 0.2 μM each of the outer primers, 1.6 mM dNTPs, 1 M betaine (Sigma), 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH4)2SO4, 4 mM MgSO4, 1.0 mL/L Triton X-100, 8 U of the Bst DNA polymerase large fragment (New England Biolabs), and 0.25 mg/L ethidium bromide. Denaturation of plasmid DNA (0.2 ng/mL in Tris-EDTA, pH 7.5) was performed at 95 °C for 5 min followed quickly by placement on ice for 5 min. Using 0.7% agarose gel electrophoresis, we confirmed that a double-stranded plasmid DNA was prepared by the Plasmid Midi reagent set and that the plasmid was heat-denatured (data not shown). The mixture was incubated at 60 °C for 1 h and analyzed using the ABI PRISM 7700 sequence detection system (Perkin-Elmer Biosystems) (2). This system measures the increase in the fluorescent intensity of ethidium bromide (bound to the amplified DNA) by use of the ROX fluorescence channel. No internal control was used. A pair of inner primers was used to amplify denatured and nondenatured double-stranded plasmid DNAs, including the HBV subtype adr sequence (3), and the DNA products were scanned using the ABI PRISM 7700. The scanning data indicated that the signal was detected in both samples after 40 min (Fig. 1 , top panel). This result suggests that the LAMP reaction does not require denatured DNA template. Detection of HBV DNA by LAMP method. (Top panel), ○ and • show LAMP reaction using nondenatured and denatured plasmid DNA templates (103 molecules), respectively. (Bottom panel), ○ and • show HBV-positive and noninfective DNA, respectively. In each sample, the signal reached a plateau after a few minutes, presumably because free ethidium bromide was depleted by binding to amplified DNA. We confirmed that the amount of DNA increased after the signal reached a plateau (data not shown). ΔRn, normalized emission at 615 nm. We performed LAMP reactions using genomic DNAs extracted from five HBV DNA-positive serum samples in which the initial copy number was unknown. When we used nondenatured DNA corresponding to 4 μL of serum as template, LAMP amplification was able to detect signals after 25–35 min in five individuals (Fig. 1 , bottom panel). This result revealed that the presence of HBV virus can be detected within 1 h from a nondenatured sample. HBV is not typical because it has double- and single-stranded segments. In separate experiments, however, LAMP was performed successfully without heat denaturation for template DNAs, such as λ DNA, pBluescript II, and M13 mp18 vector DNA, and human genomic DNA (SRY gene on chromosome Y), including commercially available material (data not shown). Some of the double-stranded DNA seems to become single-stranded at high temperatures in the presence of high concentrations of betaine, a reagent that facilitates DNA strand separation because it isostabilizes DNA (4). The exact mechanism, however, is unknown. Because there is no necessity for heat denaturation of the template DNAs, LAMP could be used more easily and rapidly in clinical medicine. We thank all members of the DUG unit at Eiken Chemical for their contributions to this work.