The RT-LAMP reaction was carried out by using the Loopamp RNA amplification kit (Eiken Chemical, Tokyo, Japan). The reaction system contained 5 μl of total RNA, 40 pmol each of the primers FIP and BIP, 5 pmol each of the outer primers F3 and B3, 20 pmol of LF, 12.5 μl of 2 × Reaction Mix, 1 μl of Enzyme Mix and 1 μl of Fluorescent regent. The reaction mixture was incubated at 63°C for 45 min in a Loopamp real-time turbidimeter LA-320 (Eiken Chemical, Tokyo, Japan) and followed by heating at 80°C for 5 min to terminate the reaction. The optical density data of each reaction was real-time recorded every 6 s. The threshold of the turbidity for positive sample was defined at 0.1. The time of positivity (Tp) was determined when the turbidity value increased above the threshold.
Loopamp rna amplification kit
Manufactured by Eiken Chemical
Sourced in Japan
The Loopamp RNA amplification kit is a laboratory equipment product used for the amplification of RNA samples. It is designed to facilitate the detection and analysis of RNA molecules through a process of isothermal amplification.
Automatically generated - may contain errors
Lab products found in correlation
Fluorescent detection reagent, by Eiken Chemical (3 mentions)
Loopamp real time turbidimeter la 230, by Eiken Chemical (2 mentions)
Loopamp real time turbidimeter la 320c, by Eiken Chemical (1 mentions)
Loopamp dna amplification kit, by Eiken Chemical (1 mentions)
Qiaamp rna mini kit, by Qiagen (1 mentions)
Primerexplorer v4, by Eiken Chemical (1 mentions)
Bst dna polymerase, by New England Biolabs (1 mentions)
14 protocols using loopamp rna amplification kit
1
RT-LAMP for RNA Amplification
2
RT-LAMP Detection of Dengue Viruses
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The serotype-specific oligonucleotide primers used for RT-LAMP assay amplification of dengue viruses were designed using the Primer-Explorer V3 software based on 3′ noncoding region (NCR) (GenBank accession number: DENV-1 Western Pacific, U88535; DENV-2 New Guinea C, AF038403; DENV-3 H87, M93130, and DEN-4 China Guangzhou B5, AF289029) (Table 1 ) [6 ]. Loop primers (Loop-F and Loop-B) were designed manually. The RT-LAMP assay was performed using Loopamp RNA amplification kit (Eiken Chemical Co. Ltd., Japan). Briefly, a 25 μl reaction mixture consisted of 1.6 μM FIP and BIP primer, 0.8 μM Loop-F and Loop-B primer, 0.2 μM F3 and B3 primer, 12.5 μl of 2X reaction mixture (RM) (provided in the kit), 1 μl of Enzyme mixture (EM) (provided in the kit), 0.7 μl of sterile deionize water, 1μl of Florescent Detection reagent (FD) (provided in the kit) and 1 μl of template RNA. Single tube RT-LAMP was conducted by adding all primer sets in a single reaction mixture. The reaction mixture was carried out in a Loopamp real-time turbidimeter (LA-320, Teramecs, Co., Ltd., Japan). To prevent cross-contamination, different sets of pipettes and different work areas were designated for template preparation, reaction mixture preparation and DNA amplification. All experiments were performed in duplicate at least 2 times.
3
Optimizing RT-LAMP Reaction Conditions
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The RT-LAMP reaction was conducted by incubating a total reaction mixture of 25 μl at 65°C for 60 min. A homemade mixture (Table S1) and two commercial mixtures (Loopamp RNA amplification kit (Eiken Chemical Co. Ltd., Japan) and Isothermal Master Mix (IMM kit) (Optigene Ltd., England)) were used. In order to optimize the RT-LAMP mixtures, different concentrations of dNTPs and primers were tested as follows: dNTPs from 0.2 to 1.4 mM, FIP and BIP (inner primers) from 20 to 40 pmole, and F3 and B3 (outer primers) from 5 to 10 pmole.
4
RT-LAMP Assay for Viral RNA Detection
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The RT-LAMP reaction was carried out in 25 μL reaction total volume using the Loopamp RNA amplification kit (Eiken Chemical Company Limited, Japan) containing 40 pmol of each of the inner primers FIP and BIP, 5 pmol each of the outer primers F3 and B3, 12.5 μL reaction mix. The reaction liquid was mixed with 1 μL enzyme mixture and 1 μL fluorescent detection regent. The reaction mixture was incubated at 63 °C for 45 min in a Loopamp real-time turbidimeter LAC320 (Eiken Chemical Company Limited, Japan) or a 63 °C water bath, followed by heating at 80 °C for 5 min to terminate the reaction.
For real-time monitoring positive result, a sample having Tp value of ≤45 min and turbidity above the threshold value of ≥0.1 was considered positive. Visualization results were completed by adding 1 μL fluorescent detection reagent Calcein (FDR, Eiken Chemical Company Limited, Japan). Orange changes to chartreuse fluorescence were regarded as positive reaction.
Sensitivity of the RT-LAMP assay was analyzed using 10-fold serial dilutions of viral RNA. The final concentrations of viral RNA were from 2.97 × 107 copies to 2.97 × 100 copies per reaction mix. Specificity of the assay was evaluated by cross reactivity tests with virus belonging to Picornaviridae and several other viral pathogens that may cause diarrhea and reproductive failure.
For real-time monitoring positive result, a sample having Tp value of ≤45 min and turbidity above the threshold value of ≥0.1 was considered positive. Visualization results were completed by adding 1 μL fluorescent detection reagent Calcein (FDR, Eiken Chemical Company Limited, Japan). Orange changes to chartreuse fluorescence were regarded as positive reaction.
Sensitivity of the RT-LAMP assay was analyzed using 10-fold serial dilutions of viral RNA. The final concentrations of viral RNA were from 2.97 × 107 copies to 2.97 × 100 copies per reaction mix. Specificity of the assay was evaluated by cross reactivity tests with virus belonging to Picornaviridae and several other viral pathogens that may cause diarrhea and reproductive failure.
5
Rapid RT-LAMP Detection of Avian Influenza
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Total nucleic acid extracts were subjected to reverse transcription loop–mediated isothermal amplification (RT-LAMP) (Eiken Chemical Co., Ltd., Tokyo, Japan) to detect viral RNA. RT-LAMP has been previously applied to detect AIV
in the fecal material of migratory birds [26 (link), 39 (link)]; the reported detection limit of RT-LAMP for fecal material is 102.5 copies [39 (link)]. For samples from 2008 and 2009, 5 µl of extracted total nucleic acids, the Loopamp RNA Amplification Kit (Eiken Chemical Co., Ltd.) and the primer set provided by Eiken
Chemical Co., Ltd. were used for the RT-LAMP reaction following the manufacturer’s instructions. For samples from 2010 to 2015, 5 µl of extracted total nucleic acid and the Loopamp AIV detection kit (Eiken
Chemical Co., Ltd.) were used. A LA-320C Loopamp Real-time turbidimeter (Eiken Chemical Co., Ltd.) was used for the RT-LAMP reaction. The threshold value for viral RNA detection was set at 0.05. Virus isolation from RT-LAMP
positive samples was conducted at reference laboratories designated by the Ministry of Environment.
in the fecal material of migratory birds [26 (link), 39 (link)]; the reported detection limit of RT-LAMP for fecal material is 102.5 copies [39 (link)]. For samples from 2008 and 2009, 5 µl of extracted total nucleic acids, the Loopamp RNA Amplification Kit (Eiken Chemical Co., Ltd.) and the primer set provided by Eiken
Chemical Co., Ltd. were used for the RT-LAMP reaction following the manufacturer’s instructions. For samples from 2010 to 2015, 5 µl of extracted total nucleic acid and the Loopamp AIV detection kit (Eiken
Chemical Co., Ltd.) were used. A LA-320C Loopamp Real-time turbidimeter (Eiken Chemical Co., Ltd.) was used for the RT-LAMP reaction. The threshold value for viral RNA detection was set at 0.05. Virus isolation from RT-LAMP
positive samples was conducted at reference laboratories designated by the Ministry of Environment.
6
RT-LAMP Assay for Rapid RNA Detection
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The RT-LAMP reaction was prepared using the Loopamp RNA Amplification Kit (Eiken Chemical Co. Ltd., Japan). Each RT-LAMP reaction (25 μl) was added with the inner primers (20 pmol each), outer primers (2.5 pmol each), loop primer (20 pmol each), Fluorescent Detection Reagent (Eiken Chemical Co. Ltd., Japan; 1 μl), and the eluted RNA (5 μl). The RT-LAMP were performed using LA-500 Loopamp real-time turbidimeter (Eiken Chemical Co. Ltd., Japan) according to the following conditions: 90 min at 63 °C followed by 5 min of assay inactivation at 80 °C. The turbidity of RT-LAMP reaction was measured at 650 nm every 6 s. The threshold time (Tt) value was recorded when the turbidity crossed the threshold cut-off value at 0.07 absorbance units [48 (link), 49 (link)].
7
RT-LAMP Assay for Rapid RNA Detection
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A Loopamp RNA amplification kit (Eiken Chemical Co., Ltd., Tokyo, Japan) was used to perform the RT-LAMP reaction. The reaction volume of 25 μL contained 2 × Reaction Mix 12.5 μL, Enzyme Mix (a mix of Bst DNA polymerase and AMV reverse transcriptase) 1.0 μL, primers FIP and BIP (40 pmol), primers F3 and B3 (5 pmol), primers LF and/or LB (20 pmol), fluorescent detection reagent 1 μL (when needed), and template RNA 2 μL. The mixture was incubated at 63°C for 60 min. The process was monitored using a Loopamp Real-time Turbidimeter (LA-230; Eiken Chemical Co., Ltd., Tochigi, Japan). Turbidity readings at an optical density at 650 nm were obtained every 6 seconds, and the reaction was considered positive when the turbidity values were >0.1. For visual observation, 1 μL of fluorescent calcein was added to the mixture, and a colour change from orange to green was observed by the naked eye for a positive reaction.
8
Isothermal Amplification of RNA Targets
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Reverse transcription loop-mediated isothermal amplification reactions were performed using a Loopamp RNA amplification kit (Eiken Chemical Co., Ltd., Tokyo, Japan) in a volume of 25 μL according to the manufacturer’s protocol. Each reaction included 80 pmol of FIP and BIP, 40 pmol of LB, 10 pmol of F3 and B3, and 2 μL template RNA. The reaction was carried out at 61°C for 60–80 min in dry bath incubators.
Reverse transcription loop-mediated isothermal amplification amplified products were detected by turbidity monitoring as well as visual observation. To assess turbidity, the amount of white magnesium phosphate precipitate produced during the LAMP reaction process was monitored using a Loopamp Real-time Turbidimeter (LA-230; Eiken Chemical Co., Ltd., Tochigi, Japan) recording the reaction curves at 650 nm every 6 s with magnesium ion (Mg2+) in the reaction buffer (Mori et al., 2001 (link)). For visual inspection, tubes containing 1 μl of fluorescent calcein were observed by the naked eye and photographed under natural light or UV light at 365 nm. The color changed from orange to green for a positive reaction, while the negative control remained orange.
Reverse transcription loop-mediated isothermal amplification amplified products were detected by turbidity monitoring as well as visual observation. To assess turbidity, the amount of white magnesium phosphate precipitate produced during the LAMP reaction process was monitored using a Loopamp Real-time Turbidimeter (LA-230; Eiken Chemical Co., Ltd., Tochigi, Japan) recording the reaction curves at 650 nm every 6 s with magnesium ion (Mg2+) in the reaction buffer (Mori et al., 2001 (link)). For visual inspection, tubes containing 1 μl of fluorescent calcein were observed by the naked eye and photographed under natural light or UV light at 365 nm. The color changed from orange to green for a positive reaction, while the negative control remained orange.
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
Dengue Diagnosis: Multimodal Approach
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Approximately 5–6 ml of blood were collected aseptically to EDTA containers and plasma was separated within 2 h and stored at − 80 °C in 2–3, 1 ml aliquots, one of which was transported to the laboratory at Stanford University, USA for RT-qPCR. NS1 test and the RT-LAMP test were performed at the laboratory at Department of Parasitology, Faculty of Medicine, University of Colombo, Sri Lanka.
In this study, dengue was diagnosed by NS1 antigen testing on admission (one step SD Bioline dengue NS 1 antigen test, Alere SD, USA, 88.65% sensitivity and 98.75% specificity up to day 9 of fever) [15 (link)] and by RT-qPCR as described by Waggoner et al. previously [16 (link)] with a Ct value cut-off of 38.5 [17 (link)]. RT-qPCR also enabled serotyping but as it was done by batch processing for cost-effectiveness, in most cases the diagnosis was made retrospectively after the patient was discharged. For RT-LAMP assay, genomic viral RNA was extracted from 140ul of patient plasma samples using QIAamp RNA mini kit (Qiagen, Germany) and the assay was performed in duplicates using Loopamp RNA amplification kit (Eiken Chemical Co. Ltd., Japan) with the primers previously published by Lau et al. [18 (link)] using 1ul of template RNA in each reaction mix [18 (link)]. Full description of assay methods is given in Additional file2 .
In this study, dengue was diagnosed by NS1 antigen testing on admission (one step SD Bioline dengue NS 1 antigen test, Alere SD, USA, 88.65% sensitivity and 98.75% specificity up to day 9 of fever) [15 (link)] and by RT-qPCR as described by Waggoner et al. previously [16 (link)] with a Ct value cut-off of 38.5 [17 (link)]. RT-qPCR also enabled serotyping but as it was done by batch processing for cost-effectiveness, in most cases the diagnosis was made retrospectively after the patient was discharged. For RT-LAMP assay, genomic viral RNA was extracted from 140ul of patient plasma samples using QIAamp RNA mini kit (Qiagen, Germany) and the assay was performed in duplicates using Loopamp RNA amplification kit (Eiken Chemical Co. Ltd., Japan) with the primers previously published by Lau et al. [18 (link)] using 1ul of template RNA in each reaction mix [18 (link)]. Full description of assay methods is given in Additional file
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