The Experts below are selected from a list of 6756 Experts worldwide ranked by ideXlab platform
Ken-ichi Hanaki - One of the best experts on this subject based on the ideXlab platform.
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Detection of murine norovirus by reverse transcription Loop-Mediated Isothermal Amplification.
Journal of Virological Methods, 2014Co-Authors: Ken-ichi Hanaki, Fumio Ike, Ayako Kajita, Wataru Yasuno, Misato Yanagiba, Motoki Goto, Kouji Sakai, Yasushi Ami, Shigeru KyuwaAbstract:Abstract Murine norovirus (MNV) has considerable genetical and biological diversity and is recognized worldwide as the most common contaminant in laboratory mouse colonies. This study developed a reverse transcription Loop-Mediated Isothermal Amplification (RT-LAMP) method with the potential to detect a broad range of MNV. RT-LAMP, using a set of five primers containing mixed bases, obtained results under Isothermal conditions at 62 °C for 90 min. Sensitivity of RT-LAMP was 50-fold less than that of two-step TaqMan real-time reverse transcription-polymerase chain reaction (TaqMan RT-PCR). Diagnostic performance of RT-LAMP on RNA extracted from mouse fecal specimens was compared with TaqMan RT-PCR and nested RT-PCR. MNV was detected in 54 of 120 mouse fecal specimens by RT-LAMP, and RT-LAMP had an estimated sensitivity and specificity of 96.4% and 100% compared with TaqMan RT-PCR, and 94.7% and 100% compared with nested RT-PCR. RT-LAMP, which does not require expensive instruments, might be useful for the screening of mice actively or persistently infected with MNV.
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colorimetric detection of loop mediated Isothermal Amplification reaction by using hydroxy naphthol blue
BioTechniques, 2009Co-Authors: Motoki Goto, Eiichi Honda, Atsuo Ogura, Akio Nomoto, Ken-ichi HanakiAbstract: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.
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colorimetric detection of loop mediated Isothermal Amplification reaction by using hydroxy naphthol blue
BioTechniques, 2009Co-Authors: Motoki Goto, Eiichi Honda, Atsuo Ogura, Akio Nomoto, Ken-ichi HanakiAbstract: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...
Xingyu Jiang - One of the best experts on this subject based on the ideXlab platform.
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uracil dna glycosylase assisted loop mediated Isothermal Amplification for detection of bacteria from urine samples with reduced contamination
Analyst, 2020Co-Authors: Yingmin Zeng, Meiling Liu, Yong Xia, Xingyu JiangAbstract:Loop-Mediated Isothermal Amplification (LAMP) is a useful molecular biology technology for analytical applications, but it is prone to contamination because of escaped aerosols, leading to false positive results. This report establishes an integrated, rapid, and accurate method to detect bacteria in urine samples by incorporating uracil-DNA-glycosylase (UDG) into real-time Loop-Mediated Isothermal Amplification (RT-LAMP). To do this, nucleic acids from five clinically important uropathogens, including Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Proteus mirabilis, and Enterococcus faecalis, were directly captured and concentrated using Flinders Technology Associates (FTA) Elute cards. Following elution, the extracted DNAs were specifically amplified with sets of LAMP primers. The added UDG in the modified LAMP reaction excises uracils from previously amplified products or contaminants and generates apyrimidinic (AP) sites, reducing false positive rates. This UDG-assisted RT LAMP strategy was able to degrade carryover contaminants to as little as 1 femtogram (10-15 g). The assay showed a limit of detection of 104 CFU mL-1 with a sensitivity of 94.1% and a specificity of 95.0%. Both the sensitivity and specificity were improved compared to LAMP carried out without UDG. Our results indicate that the UDG-assisted RT LAMP is of great potential for rapid and precise analysis of nucleic acids in real applications.
Tsugunori Notomi - One of the best experts on this subject based on the ideXlab platform.
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loop mediated Isothermal Amplification lamp expansion of its practical application as a tool to achieve universal health coverage
Journal of Infection and Chemotherapy, 2020Co-Authors: Yasuyoshi Mori, Tsugunori NotomiAbstract:Since its invention in 2000, Loop-Mediated Isothermal Amplification (LAMP) has attracted great interest from researchers and has been used as a simple and rapid diagnostic tool for detection of infectious and non-infectious diseases. Here we review the recent circumstances and outcomes of these applications of LAMP to show the potential of LAMP as a tool for achieving universal health coverage (UHC). A future application of LAMP, such as in an automated multiplex format, is also discussed.
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mitochondrial dna targets increase sensitivity of malaria detection using loop mediated Isothermal Amplification
Journal of Clinical Microbiology, 2010Co-Authors: Spencer D Polley, Tsugunori Notomi, Iveth J Gonzalez, Yasuyoshi Mori, Julie Watson, Mark D Perkins, Peter L Chiodini, Colin J SutherlandAbstract:Loop-Mediated Isothermal Amplification (LAMP) of DNA offers the ability to detect very small quantities of pathogen DNA following minimal tissue sample processing and is thus an attractive methodology for point-of-care diagnostics. Previous attempts to diagnose malaria by the use of blood samples and LAMP have targeted the parasite small-subunit rRNA gene, with a resultant sensitivity for Plasmodium falciparum of around 100 parasites per μl. Here we describe the use of mitochondrial targets for LAMP-based detection of any Plasmodium genus parasite and of P. falciparum specifically. These new targets allow routine Amplification from samples containing as few as five parasites per μl of blood. Amplification is complete within 30 to 40 min and is assessed by real-time turbidimetry, thereby offering rapid diagnosis with greater sensitivity than is achieved by the most skilled microscopist or antigen detection using lateral flow immunoassays.
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accelerated reaction by loop mediated Isothermal Amplification using loop primers
Molecular and Cellular Probes, 2002Co-Authors: Kentaro Nagamine, Tetsu Hase, Tsugunori NotomiAbstract:Abstract Loop-Mediated Isothermal Amplification (LAMP) is a novel nucleic acid Amplification method that amplifies DNA with high specificity, efficiency and rapidity under Isothermal conditions using a set of four specially designed primers and a DNA polymerase with strand displacement activity. We have developed a method that accelerates the LAMP reaction by using additional primers, termed loop primers. Loop primers hybridize to the stem-loops, except for the loops that are hybridized by the inner primers, and prime strand displacement DNA synthesis. Although both inner and loop primers react via the loops, they do so by different mechanisms. The LAMP method presented here uses loop primers to achieve reaction times of less than half that of the original LAMP method. Since the total time of analysis including detection is less than 1 h, this new method should facilitate genetic analysis, including genetic diagnosis in the clinical laboratory.
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isolation of single stranded dna from loop mediated Isothermal Amplification products
Biochemical and Biophysical Research Communications, 2002Co-Authors: Kentaro Nagamine, Yoko Kuzuhara, Tsugunori NotomiAbstract:Loop-Mediated Isothermal Amplification (LAMP), in which a specific DNA sequence can be directly amplified under Isothermal conditions, yields DNA in large quantities of more than 500 microg/ml. We have developed a method to isolate single-stranded DNA fragments from LAMP products that are stem-loop DNAs with several inverted repeats of the target DNA. This method requires the TspRI restriction enzyme, a primer hybridized to the 3' overhanging sequence at its cleavage site, and a DNA polymerase with strand displacement activity. The LAMP products are digested with TspRI and are then extended using the primer, producing the strand-specific DNA fragments. All processes, from LAMP reaction to primer extension, can be carried out at the same temperature. The use of strand-specific DNA would be conducive for detection by hybridization technique such as DNA microarrays.
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detection of loop mediated Isothermal Amplification reaction by turbidity derived from magnesium pyrophosphate formation
Biochemical and Biophysical Research Communications, 2001Co-Authors: Yasuyoshi Mori, Kentaro Nagamine, Norihiro Tomita, Tsugunori NotomiAbstract:The Loop-Mediated Isothermal Amplification (LAMP) is a novel nucleic acid Amplification method that uses only one type of enzyme. One of the characteristics of the LAMP method is its ability to synthesize extremely large amount of DNA. Accordingly, a large amount of by-product, pyrophosphate ion, is produced, yielding white precipitate of magnesium pyrophosphate in the reaction mixture. Judging the presence or absence of this white precipitate allows easy distinction of whether nucleic acid was amplified by the LAMP method. Since an increase in the turbidity of the reaction mixture according to the production of precipitate correlates with the amount of DNA synthesized, real-time monitoring of the LAMP reaction was achieved by real-time measurement of turbidity.
Motoki Goto - One of the best experts on this subject based on the ideXlab platform.
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Detection of murine norovirus by reverse transcription Loop-Mediated Isothermal Amplification.
Journal of Virological Methods, 2014Co-Authors: Ken-ichi Hanaki, Fumio Ike, Ayako Kajita, Wataru Yasuno, Misato Yanagiba, Motoki Goto, Kouji Sakai, Yasushi Ami, Shigeru KyuwaAbstract:Abstract Murine norovirus (MNV) has considerable genetical and biological diversity and is recognized worldwide as the most common contaminant in laboratory mouse colonies. This study developed a reverse transcription Loop-Mediated Isothermal Amplification (RT-LAMP) method with the potential to detect a broad range of MNV. RT-LAMP, using a set of five primers containing mixed bases, obtained results under Isothermal conditions at 62 °C for 90 min. Sensitivity of RT-LAMP was 50-fold less than that of two-step TaqMan real-time reverse transcription-polymerase chain reaction (TaqMan RT-PCR). Diagnostic performance of RT-LAMP on RNA extracted from mouse fecal specimens was compared with TaqMan RT-PCR and nested RT-PCR. MNV was detected in 54 of 120 mouse fecal specimens by RT-LAMP, and RT-LAMP had an estimated sensitivity and specificity of 96.4% and 100% compared with TaqMan RT-PCR, and 94.7% and 100% compared with nested RT-PCR. RT-LAMP, which does not require expensive instruments, might be useful for the screening of mice actively or persistently infected with MNV.
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colorimetric detection of loop mediated Isothermal Amplification reaction by using hydroxy naphthol blue
BioTechniques, 2009Co-Authors: Motoki Goto, Eiichi Honda, Atsuo Ogura, Akio Nomoto, Ken-ichi HanakiAbstract: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.
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colorimetric detection of loop mediated Isothermal Amplification reaction by using hydroxy naphthol blue
BioTechniques, 2009Co-Authors: Motoki Goto, Eiichi Honda, Atsuo Ogura, Akio Nomoto, Ken-ichi HanakiAbstract: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...
Jun Peng - One of the best experts on this subject based on the ideXlab platform.
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detection of cucumber mosaic virus isolates from banana by one step reverse transcription loop mediated Isothermal Amplification
Archives of Virology, 2012Co-Authors: Jun Peng, Minjing Shi, Zihao Xia, Junsheng Huang, Zaifeng FanAbstract:Cucumber mosaic virus (CMV) is one of the most devastating threats to the banana industry. A single-tube, one-step reverse transcription Loop-Mediated Isothermal Amplification (RT-LAMP) assay was developed for the rapid detection of CMV-infected banana and plantain (Musa spp.). The reaction was performed in a single tube at 63 °C for 90 min using a real-time turbidimeter, with an improved closed-tube visual detection system in which fluorescent dye was added to the inside of the lid prior to Amplification. This RT-LAMP assay is an alternative method for the rapid detection of CMV in banana plants and tissue culture materials.
