All of the oligomers were commercially synthesized and purified by Tsingke (Beijing, China). The Loopamp DNA amplification kits and Loopamp™ Fluorescent Detection Reagent (FD) were purchased from Eiken Chemical Co. Ltd. (Beijing, China). The DNA Extraction Kits (QIAamp DNA minikits) and QIAquick Gel Extraction Kit (QIAGEN, Hidlen, Germany) were purchased from Qiagen (Beijing, China).
Loopamp dna amplification kit
Manufactured by Eiken Chemical
Sourced in Japan
The Loopamp DNA amplification kit is a laboratory equipment designed for the amplification of DNA samples. It utilizes a DNA amplification technique to rapidly and efficiently replicate target DNA sequences. The core function of the kit is to provide a reliable and consistent method for amplifying DNA samples for various analytical and research applications.
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Lab products found in correlation
Realoop 30, by Eiken Chemical (2 mentions)
Loopamp fluorescent detection reagent fd, by Eiken Chemical (1 mentions)
Qiaquick gel extraction kit, by Qiagen (1 mentions)
Digital gel documentation system, by Bio-Rad (1 mentions)
Veriti thermal cycler, by Thermo Fisher Scientific (1 mentions)
Loopamp rna amplification kit, by Eiken Chemical (1 mentions)
Loopamp exia, by Eiken Chemical (1 mentions)
Thermopol buffer, by New England Biolabs (1 mentions)
Loopamp realtime turbidimeter, by Eiken Chemical (1 mentions)
Mgso4, by New England Biolabs (1 mentions)
Betaine, by Merck Group (1 mentions)
Loopamp real time turbidimeter la 320c, by Eiken Chemical (1 mentions)
Loopamp real time turbidimeter la 230, by Eiken Chemical (1 mentions)
Geneamp pcr system 9700, by Thermo Fisher Scientific (1 mentions)
Rnase inhibitor, by Thermo Fisher Scientific (1 mentions)
Syto 9 green, by Thermo Fisher Scientific (1 mentions)
Qiaamp kit, by Qiagen (1 mentions)
Evagreen, by Biotium (1 mentions)
Ethanol, by Merck Group (1 mentions)
Acetone, by Merck Group (1 mentions)
20 protocols using loopamp dna amplification kit
1
Oligomer Synthesis and Purification
2
LAMP Assay for Blood Sample Analysis
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LAMP assays were performed on the 319 blood samples using a protocol reported previously [25] (link). The primers used are listed in Table 1 . Briefly, reaction mixtures were prepared with Loopamp DNA amplification kits (Eiken Chemical Co., Ltd.; Japan) as follows: 2.4 µM of both FIP and BIP primers, 0.2 µM of both F3 and B3c primers, 0.8 µM of LPF and LPB, 12.5 µL of 2× reaction mixture (40 mM Tris-HCl [pH 8.8], 20 mM KCl, 16 mM MgSO4, 20 mM (NH4)2SO4, 0.2% Tween 20, 1.6 M betaine, and 2.8 mM of each deoxyribonucleoside triphosphate), Bst DNA polymerase, and 5 µL of DNA sample. LAMP was performed at 60°C in a water bath for 60 min, and visibly turbid samples were considered positive, in a single blind manner. LAMP reaction mixes were stained with SYBR green I dye (Eugene, USA), and positive samples were identified with the naked eye under an ultraviolet transilluminator or using a handheld black light.
3
Colorimetric RT-LAMP with Dried Crystal Violet
4
LAMP Assays for Plasmodium Detection
5
Colorimetric LAMP Assay with LCV
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LCV solution containing 0.5 mM crystal violet (CV), 30 mM sodium sulfite, and 5 mM β-cyclodextrin was prepared and stored at −20 °C until use. For field use, LCV can be lyophilized for long shelf life at room-temperature (13 (link)). The first-stage RPA mix was carried out as described above. The second-stage LAMP reaction was performed with a Loopamp DNA amplification kit (Eiken Chemical Co., Ltd., Japan) that, in contrast to the Optigene kit, does not include fluorescent dye. To ease hand-mixing, we used 70 μL LAMP volume. The LAMP reaction mix included: 1.6 μM FIP and BIP, 0.8 μM LF and LB; 1 μL Bst DNA polymerase (provided in Loopamp DNA amplification kit, http://loopamp.eiken.co.jp/e/products/dna/ ); and 5.5 μL LCV solution. The reaction buffer was incubated at 63°C for up to 40 min. The LCV color change was observed at the end of the incubation process by naked eye and, if desired, it can be recorded with a smartphone.
6
Isothermal DNA Amplification Using LAMP
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The isothermal DNA amplification reaction was performed using the loopamp DNA amplification kit, following the protocol from the manufacturer (Eiken Chemical Co., Tokyo, Japan). Each LAMP reaction included the following reagents: 2 µL of genomic DNA, 12.5 µL of reaction mixture, 1 µL of primer mixture (F3, B3, FIP, and BIP), 1 µL of Bst DNA polymerase, and 8.5 µL of molecular grade water. The prepared mixture was then incubated under an isothermal condition (65°C) for 40 min in a real-time PCR machine (Veriti Thermal Cycler, Applied Biosystems). The resultant LAMP products were analyzed by 1.7% gel electrophoresis, visualized with a UV transilluminator, and the images were recorded with a digital gel documentation system (Bio-Rad).
7
Automated DNA Detection with Graphite Heating
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In addition to implementing cell lysis using micro-carbon-activated heating for fully automated DNA detection on the LOD, DNA amplification was a main objective of the present study. The Loopamp DNA Amplification Kit (Eiken Chemical Co., Ltd., Tokyo, Japan) and a graphite sheet (graphite depth: 0.1 mm, GENetech, Seoul, Korea) were used because the LAMP process was performed at 60 °C. Figure 7 shows the location of the graphite sheet on the disc. To illustrate its function, a side-view schematic of the graphite is also presented.
Graphite is a substance with a high heat of fusion, melting, and solidification at a certain temperature; thus, it can store and release large amounts of energy. When graphite is transformed into sheet form (hereinafter referred to as a “G-sheet”) with a thickness of a few micrometers, its state becomes very sensitive to temperature changes. For a G-sheet, the phase change occurs at a temperature of 60 °C. In this study, the IR camera measured the temperature distribution as a 2D image map.
Graphite is a substance with a high heat of fusion, melting, and solidification at a certain temperature; thus, it can store and release large amounts of energy. When graphite is transformed into sheet form (hereinafter referred to as a “G-sheet”) with a thickness of a few micrometers, its state becomes very sensitive to temperature changes. For a G-sheet, the phase change occurs at a temperature of 60 °C. In this study, the IR camera measured the temperature distribution as a 2D image map.
8
Molecular Detection of Vibrio Pathogenicity Genes
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tdh gene: Loop-mediated isothermal amplification assay was applied to detect tdh gene using a Loopamp DNA amplification kit (Eiken Chemical Co., Ltd.,
Tokyo, Japan) as described by Yamazaki et al. [25 (link)]. The incubation was carried out in a Loop realtime tubidimeter (Realoop-30, Eiken
Chemical) at 65°C for 60 min, following by 80°C for 2 min. A reaction was considered positive as the turbidity reached 0.1 within 60 min.
trh gene: PCR assay for trh gene examination was performed using the protocol described previously [2 (link)]. PCR amplified
products were checked in 1.5% agarose gels by electrophoresis. After that, the gel was stained with ethidium bromide (0.5 mg/ml) and photographed under a UV
transilluminator.
Tokyo, Japan) as described by Yamazaki et al. [25 (link)]. The incubation was carried out in a Loop realtime tubidimeter (Realoop-30, Eiken
Chemical) at 65°C for 60 min, following by 80°C for 2 min. A reaction was considered positive as the turbidity reached 0.1 within 60 min.
trh gene: PCR assay for trh gene examination was performed using the protocol described previously [2 (link)]. PCR amplified
products were checked in 1.5% agarose gels by electrophoresis. After that, the gel was stained with ethidium bromide (0.5 mg/ml) and photographed under a UV
transilluminator.
9
LAMP Primer Design and Amplification Protocol
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All LAMP primer sequences and amplification temperatures can be found in S1 Table . Primers were purchased from Eurofins MWG Operon. Reactions were carried out using a LoopAmp DNA Amplification Kit and a LoopAmp RNA Amplification Kit (Eiken Chemical Co., Ltd. Tokyo, Japan). For all reactions the following amounts of primers were used: 5 pmol each of F3 and B3, 20 pmol each LF and LB, and 40 pmol each of FIP and BIP. Positive and negative controls were included in each run. For primers designed in-house, LAMP PrimerExplorer V4 software was used (http://primerexplorer.jp/elamp4.0.0/index.html ; Eiken Chemical Co., Ltd. Tokyo, Japan).
10
LAMP Assay for Pneumocystis jirovecii
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The LAMP assay was conducted with the primers F3, B3, FIP, BIP, FL, and BL, as previously described [15 (link), 16 (link)]. The LAMP reaction was performed with a Loopamp DNA amplification kit® (Eiken Chemical, Tokyo, Japan) using reaction mixtures composed of 40 pmol each of primers FIP and BIP, 5 pmol each of primers F3 and B3, 20 pmol each of primers FL and BL; 12.5 μL of 2 × reaction mixture; 1 μL of Bst deoxyribonucleic acid (DNA) polymerase; 2 μL of DNA sample; and distilled water up to a final volume of 25 μL. The mixtures were incubated at 61 °C for 60 min (Realoop-30; Eiken Chemical) and then heated at 80 °C for 2 min to terminate the reaction. The positive control solution for Pneumocystis jirovecii plasmid DNA (6 × 106 copies per tube) [15 (link)] and negative control solutions of Tris–EDTA buffer were also prepared with each assay. LAMP products were detected by real-time turbidity detection using a Loopamp EXIA® (Eiken Chemical, Tokyo, Japan).
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