Genie 2

Manufactured by OptiGene

Sourced in United Kingdom, France, Germany, United States

The Genie II is a compact, real-time isothermal amplification system designed for rapid and sensitive detection of target nucleic acid sequences. It provides a simple and efficient platform for various isothermal amplification techniques, including LAMP, HDA, and others. The Genie II system is capable of detecting and analyzing amplification results in real-time through fluorescence detection.

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Lab products found in correlation

Isothermal master mix, by OptiGene (5 mentions) Genie 3, by OptiGene (4 mentions) Iso 001, by OptiGene (3 mentions) Genie explorer v0, by OptiGene (3 mentions) Isothermal master mix iso 001, by OptiGene (3 mentions) Qubit 3.0 fluorometer, by Thermo Fisher Scientific (3 mentions) Amplifyrp xrt, by Agdia (2 mentions) Gspssd isothermal mastermix iso 001, by OptiGene (2 mentions) Lamp designer v 1, by Premier Biosoft (2 mentions) Bst 2.0 dna polymerase, by New England Biolabs (2 mentions) Ese quant tubescanner, by Qiagen (2 mentions) Transcriptor reverse transcriptase, by New England Biolabs (2 mentions) Gspssd 2.0 isothermal mastermix, by OptiGene (2 mentions) Amv reverse transcriptase, by New England Biolabs (1 mentions) Stratagene mx3005p, by Agilent Technologies (1 mentions) Lyse n lamp master mix, by OptiGene (1 mentions) Genie 3 system, by OptiGene (1 mentions) Twistamp liquid exo kit, by Twist Bioscience (1 mentions) Pcr grade water, by Qiagen (1 mentions) Gel doc, by Bio-Rad (1 mentions)

31 protocols using genie 2

Rapid PPRV Detection via RT-LAMP

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RT-LAMP was performed in a total reaction mixture of 25 µL containing 15 µL isothermal master mix ISO-001 (OptiGene Ltd., Horsham, UK), optimised primer concentrations as per Table 2, 2 U AMV reverse transcriptase (New England Biolabs), 5 µL template RNA and made up to volume with nuclease-free water. RT-LAMP reactions were incubated at 65 °C for 60 min on either a Stratagene Mx3005p (Agilent Technologies, Stockport, UK) or Genie II (OptiGene Ltd.). For positive RT-LAMP reactions, the time to positivity (T_p) was defined when reactions reached a fluorescence threshold increase of δR 1000.
To confirm that amplicons were PPRV-specific, annealing analysis was performed on RT-LAMP products using the Genie II (OptiGene Ltd.). LAMP products were heated to 98 °C for 1 min, then cooled to 80 °C (ramping at 0.05 °C/s). Anneal temperature (T_a) calculations were automated using Genie Explorer v0.2.1.1 software (OptiGene Ltd.). Samples were considered positive if amplification had occurred and the LAMP product annealed in the PPRV amplicon-specific temperature range of 83.1–85.1 °C (mean T_a 84.1 °C +/- 1 °C of 41 PPRV positive RT-LAMP reactions).

Mahapatra M., Howson E., Fowler V., Batten C., Flannery J., Selvaraj M, & Parida S. (2019). Rapid Detection of Peste des Petits Ruminants Virus (PPRV) Nucleic Acid Using a Novel Low-Cost Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) Assay for Future Use in Nascent PPR Eradication Programme. Viruses, 11(8), 699.

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Optimizing LAMP Reaction Conditions

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The reaction temperature was optimized using a block gradient from 60 to 68°C at 0.1°C intervals followed by an annealing step of 98–80°C, ramping at 0.05°C per second. LAMP reactions contained 15 μL of the fast isothermal master mix (ISO‐004, OptiGene), 5 pmol of each primer F3 and B3, 10 pmol of each Loop‐F and Loop‐R, 20 pmol of FIP and BIP, either 10⁵ copies of the recombinant plasmid or 5 μl of the extracted DNA and nuclease‐free water to a final volume of 25 μl.
When DNA was extracted using KOH (as described below), the isothermal Lyse'n’LAMP master mix (ISO‐001LNl, OptiGene) was used instead.
Isothermal amplification was performed either in a Genie® II or a Genie® III system (OptiGene) for real‐time monitoring of the LAMP amplification. The amplification ratio measured as the change of fluorescence over time and expressed as the time of positivity (Tp), and the amplicon annealing temperature were analysed using a Genie® II or a Genie® III software (OptiGene).

Cano I., McCullough R., Mulhearn B., Gunning S., Waine A., Joiner C, & Paley R. (2020). Non‐lethal loop‐mediated isothermal amplification assay as a point‐of‐care diagnostics tool for Neoparamoeba perurans, the causative agent of amoebic gill disease. Journal of Fish Diseases, 43(7), 779-790.

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Dengue Virus RT-LAMP Detection Assays

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RT-LAMP reactions were run at 64°C using either an ESE-Quant TubeScanner (QIAGEN Lake Constance GmbH, Stockach, Germany) or Genie II (Optigene, Horsham, UK), in a final reaction volume of 25 μL. The Genie II device displays the annealing curve for specificity after the reaction has finished, by melting curve analysis from 98°C to 80°C (0.05°C/s).
Four RT-LAMP assays were developed, one for each DENV serotype (S1 File). Each reaction consisted of 1x RM Trehalose, 6 mM MgSO₄, 5% polyethylene glycol (PEG), 1 μL fluorochrome dye (FD), 8 U Bst 2.0 DNA Polymerase (New England BioLabs, Hitchin, Herts, UK), 10 U Transcriptor Reverse Transcriptase (Roche) and 1 μL template (DENV RNA or H₂O as negative control). For each primer set per RT-LAMP assay, the final concentrations was as follows: 50 nM F3, 50 nM B3, 400 nM FIP, 400 nM BIP, 200 nM FLOOP, 200 nM BLOOP. Before adding the Bst 2.0 DNA Polymerase, Transcriptor Reverse Transcriptase and template, mixes were incubated at 95°C for 5 min to melt any primer multi-mers and cooled immediately on ice for 5 min. Reaction times vary for each RT-LAMP protocol, running for 45 min (DENV1), 90 min (DENV2), 75 min (DENV3) and 50 min (DENV4).
RM Trehalose, MgSO₄, PEG and FD were supplied by MAST Diagnostica GmbH.

Lopez-Jimena B., Bekaert M., Bakheit M., Frischmann S., Patel P., Simon-Loriere E., Lambrechts L., Duong V., Dussart P., Harold G., Fall C., Faye O., Sall A.A, & Weidmann M. (2018). Development and validation of four one-step real-time RT-LAMP assays for specific detection of each dengue virus serotype. PLoS Neglected Tropical Diseases, 12(5), e0006381.

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LAMP Reaction with Universal QProbe System

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LAMP reaction with the universal QProbe system was carried out with the previously described methods with slight modifications^{38 (link)}. Briefly, the LAMP reaction mixture (25 µl) contained 1.4 mM each dNTP, 20 mM Tris–HCl (pH 8.8), 10 mM KCl, 8 mM MgSO₄, 10 mM (NH₄) ₂SO₄, 0.1% Tween 20, and 0.8 M betaine, 0.2 µM each of F3 and B3 primers, 1.6 µM each of FIP and BIP primers, 0.8 µM each of Loop F and Loop B primers, 0.14 µM of UQprobe-G (J-Bio21, Tokyo, Japan), 0.4 µM of Joint DNA 1 or 2, Bst DNA polymerase (8 U) (Nippon Gene, Tokyo, Japan) and DNA (30 ng) as a template. LAMP reaction at 66 °C, as well as a subsequent melting curve analysis from 30 or 35 °C to 95 °C with a decrement of 0.2 °C per second, were performed by Genie II (OptiGene, Horsham, UK). Conventional LAMP reactions with SIX4 primer set were conducted as described in a previously report^{18 (link)}, with the Fluorescent detection reagent (Eiken Kagaku, Tokyo, Japan) as a fluorescent dye instead of UQprobe-G. Conventional LAMP reaction with SIX3 primer set mixture, which contains above-mentioned except UQprobe-G and joint DNAs was incubated at 59–66 °C for 60 min followed by 10 min of incubation at 98 °C with Genie II (OptiGene) to detect and monitor fluorescence.

Ayukawa Y., Hanyuda S., Fujita N., Komatsu K, & Arie T. (2017). Novel loop-mediated isothermal amplification (LAMP) assay with a universal QProbe can detect SNPs determining races in plant pathogenic fungi. Scientific Reports, 7, 4253.

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RT-LAMP Assay for Rapid Detection

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RT-LAMP reactions were run at 64°C for 60 min using either an ESE-Quant TubeScanner (QIAGEN Lake Constance GmbH, Stockach, Germany) or Genie II (Optigene, Horsham, UK), in a final reaction volume of 25 μL. The Genie II device allows to create an annealing curve for confirmation of amplification specificity by an additional heating and cooling step from 98°C to 80°C (0.05°C/s) for 6 min to allow the re-annealing of the amplified product.
Each reaction consisted of 1x RM Trehalose, 6 mM MgSO₄, 5% polyethylene glycol (PEG), 1 μL fluorochrome dye (FD), 0.1 μM F3, 0.1 μM B3, 0.8 μM FIP, 0.8 μM BIP, 0.4 μM FLOOP, 0.4 μM BLOOP (final concentration for each set of primers), 8 U Bst 2.0 DNA Polymerase, 10 U Transcriptor Reverse Transcriptase and 1 μL template (RNA or H₂O as negative control). Before adding Bst 2.0 DNA Polymerase, Transcriptor Reverse Transcriptase and template, mixes were incubated at 95°C for 5 min to melt any primer multimers and cooled immediately on ice for 5 min.
RM Trehalose, MgSO₄, PEG and FD were supplied by MAST Diagnostica GmbH (Reinfeld, Germany). Bst 2.0 DNA Polymerase and Transcriptor Reverse Transcriptase were obtained from New England BioLabs (Hitchin, Herts, UK) and Roche, respectively.

Lopez-Jimena B., Wehner S., Harold G., Bakheit M., Frischmann S., Bekaert M., Faye O., Sall A.A, & Weidmann M. (2018). Development of a single-tube one-step RT-LAMP assay to detect the Chikungunya virus genome. PLoS Neglected Tropical Diseases, 12(5), e0006448.

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Real-time RPA Assay for Rapid Detection

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For real-time RPA, the TwistAmp Liquid Exo kit (TwistDx, Maidenhead, UK) was used according to the manufacturer’s instructions. The optimized reaction mix consisted of 1× reaction buffer, 0.45 mM of each dNTP, 1× Probe E-mix, 600 mM of each RPA primer, 200 nM of RPA probe, 1× Core Reaction Mix, 1× Exo, and 19 nM of MgOAc in a total volume of 50 µl with 1 µl of crude extract. Reaction incubation and fluorescence signal reading were performed using fluorimeters AmplifyRP^® XRT (Agdia, Île-de-France, France) or Genie^® II (OptiGene, Horsham, UK), consisting of 45°C for 35 min. All mixtures were shaken 5 min after the start of the reaction.

Morán F., Herrero-Cervera M., Carvajal-Rojas S, & Marco-Noales E. (2023). Real-time on-site detection of the three ‘Candidatus Liberibacter’ species associated with HLB disease: a rapid and validated method. Frontiers in Plant Science, 14, 1176513.

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Evaluation of Real-Time RPA Repeatability

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The repeatability and reproducibility of real-time RPA were evaluated using 10 plant samples (473, 607, 614, 615, 623, 627, 631, 633, 635, and 641) of C. sinensis cv. Valencia, originating from Costa Rica (Alajuela), infected with relatively low CaLas titers (low Cqs values, according to the real-time qPCR test by de Chaves et al. (2023) (link). Each crude extract sample selected was analyzed using two fluorimeters: AmplifyRP^® XRT (Agdia, Île-de-France, France) and Genie^® II (OptiGene, Horsham, UK). For each instrument, three independent replicates were carried out on different days and by two different operators.

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LAMP Reaction Protocol for Isothermal Amplification

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LAMP reactions were carried out as suggested in the OptiGene Ltd. LAMP User Guides (Version 1.1, OptiGene Ltd., Horsham, UK). For each LAMP reaction, a 25-µL reaction mixture containing 15 μL of GspSSD isothermal mastermix (ISO-001) (OptiGene Ltd.), 2.5 μL primer mix, 2.5 μL PCR-grade water (Qiagen GmbH, Hilden, Germany), as well as 5 μL template, was prepared. LAMP reactions were performed using the real-time fluorometer Genie^® II (OptiGene Ltd., Horsham, UK).

Hess J., Kreitlow A., Rohn K., Hennig-Pauka I, & Abdulmawjood A. (2023). Rapid Diagnostic of Streptococcus suis in Necropsy Samples of Pigs by thrA-Based Loop-Mediated Isothermal Amplification Assay. Microorganisms, 11(10), 2447.

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Rapid LAMP Assay for Pathogen Detection

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Amplification primers (Supplementary Table S2) for LAMP were designed using LAMP Designer v. 1.12 (Premier Biosoft, Palo Alto, CA). LAMP conditions were as described for calcein detection^{43 (link)}, and a LAMP temperature of 63 °C (1 hour assay time) was used. Alternatively, the same LAMP primers were used with detection using Isothermal Detection Reagent (Prolab Diagnostics, Richmond Hill, ON, Canada) at a temperature of 63 °C for 30 minutes. After amplification, an annealing curve was generated (90 °C–75 °C at 0.05 °C/sec). Reactions were monitored in real time using a Genie II or Genie III instrument (OptiGene, Horsham, UK), and the time to positive (T_p) was reported by the instrument. For binomial (positive/negative) detection, reactions using calcein-based detection were viewed under ultraviolet light using a transilluminator (Bio-Rad Gel Doc). The 2015 samples were analyzed undiluted and diluted 1:50 with 10 mM Tris-Cl, pH 8.5 to examine the effect of co-purifying inhibitors.

Pérez-López E., Rodríguez-Martínez D., Olivier C.Y., Luna-Rodríguez M, & Dumonceaux T.J. (2017). Molecular diagnostic assays based on cpn60 UT sequences reveal the geographic distribution of subgroup 16SrXIII-(A/I)I phytoplasma in Mexico. Scientific Reports, 7, 950.

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Lamp Assay for Phytoplasma Detection

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A LAMP assay was designed to the 16S rRNA region by identifying regions of sequence suitable for primer design from sequence alignments described previously (Hodgetts et al., 2008 (link)), which included a range of phytoplasmas from diverse 16Sr groups with the addition of other publicly available sequences from phytoplasmas and a range of other bacteria. Primers for the 16S rRNA were designed without the use of software. A second FD assay was also used, designed to the 23S rRNA, and available in kit form from OptiGene Ltd (http://www.optigene.co.uk). Primers for the 16S rRNA assay were synthesized by either Integrated DNA Technologies or Eurofins MWG Operon. All LAMP reactions were performed in single tubes, 8-well strips or 96-well plates in a 25 μL reaction volume, containing 1 or 5 μL of sample DNA or 10-times diluted plant homogenate, 2× isothermal master mix (OptiGene), 0·2 μm F3 and B3 primers, 2 μm FIP and BIP primers and 1 μm F-loop and B-loop primers. LAMP reactions were performed in a GenieII (OptiGene) or in a Roche LC480 instrument. For LAMP product annealing temperature determination (Tm), the samples were heated to 98°C and then cooled to 80°C, fluorescence was detected in real-time (on the FAM channel for the Roche LC480) and the annealing temperature recorded.

Kogovšek P., Hodgetts J., Hall J., Prezelj N., Nikolić P., Mehle N., Lenarčič R., Rotter A., Dickinson M., Boonham N., Dermastia M, & Ravnikar M. (2014). LAMP assay and rapid sample preparation method for on-site detection of flavescence dorée phytoplasma in grapevine. Plant Pathology, 64(2), 286-296.

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