lamp reaction mixtures  (New England Biolabs)

Journal: medRxiv

Article Title: International Multi-site Implementation of Local Cell-Free Protein Biomanufacturing to Advance Health and Research Equity

doi: 10.1101/2025.07.25.25332228

Figure Lengend Snippet: (a) Schematic representation of the pipeline used to create the molecular diagnostic systems. Detection was carried out using real-time fluorescence or visual detection. For fluorescence measurements, amplicons were visualized by adding 1x LAMP fluorescent dye or 10 μM SYTO 9 Green Fluorescent Nucleic Acid dye if FluoroPLUM was used, with fluorescence reads every minute. For visual detection, the naked eye confirmed amplification under natural light by adding SYBR Gold Nucleic Acid Stain (diluted 1:10) to tube caps before reaction incubation, followed by mixing after incubation. A positive reaction resulted in a color change from orange to yellow, while a negative reaction remained orange. (b) End point fluorescence levels (25 minutes for all targets) for 16 pathogens, comparing in-house (blue) and commercial LAMP systems (green). Data are shown as mean ± SD, n = 3. (c) Real-time fluorescent experiments for synthetic P. falciparum DNA amplification using a conventional qPCR instrument. This representative data was obtained using reagents produced on-site in Canada. Data are shown as mean ± SD, n = 3. (d) Real-time fluorescent experiments for synthetic SARS-CoV-2 RNA amplification using a conventional qPCR instrument. This representative data was obtained using reagents produced on-site in Canada. Data are shown as mean ± SD, n = 3. (e) Reproducibility assessment of LAMP and RT-LAMP performed in Canada, Chile, Brazil, and Colombia. Independent experiments conducted by different teams across diverse settings, using a standardized protocol, confirmed the reproducibility of the results. The consistent results reinforce the feasibility of decentralized enzyme production. (f) Comparison of the low-cost, portable FluoroPLUM and a conventional high-cost qPCR machine for assessing in-house-produced LAMP reactions, with results showing comparable performance across both platforms, confirming the utility of FluoroPLUM as an affordable alternative for high-throughput measurements in low-resource settings. (g) Real-time fluorescence and visual outputs of LAMP reactions using P. falciparum synthetic DNA (2 nM) and NTC measured with the low-cost, portable FluoroPLUM. This representative data was obtained using reagents produced on-site in Canada. Data are shown as mean ± SD, n = 3. (h) Real-time fluorescence and visual outputs of LAMP reactions using L. braziliensis synthetic DNA (2 nM) and NTC measured with the low- cost, portable FluoroPLUM. This representative data was obtained using reagents produced on-site in Canada. Data are shown as mean ± SD, n = 3. (i) Deploying low-cost cell-free lysates and open-source hardware enables decentralized biomanufacturing, facilitating the implementation of diagnostic programs in resource-limited settings. (j) End point fluorescence levels (30 minutes for all targets) for B. burgdorferi , POWV, and M. tuberculosis measured using the low-cost, portable FluoroPLUM. This representative data was obtained using diagnostic reagents manufactured on-site in Algonquin Highlands, Ontario, Canada. Data are shown as mean ± SD, n = 3. Abbreviations are: (-) or NTC, non-template control; RT, room temperature; Min, minutes; HIV-1, human immunodeficiency virus 1; CHIKV, chikungunya virus; MPXV, monkeypox virus; ZIKV, Zika virus; DENV-2, dengue virus serotype 2; WNV, West Nile virus; MAYV, Mayaro virus; POWV, Powassan virus; OROV, Oropouche virus.

Article Snippet: In brief, LAMP reaction mixtures contained 1X isothermal buffer (20 mM Tris-HCl, 10 mM (NH4)2SO4, 50 mM KCl, 2 mM MgSO4, 0.1% Tween 20, pH 8.8), 4 mM MgSO 4 , 1.4 mM deoxynucleotides triphosphates (dNTPs) (NEB, N0446S), 10X primer mix, and our in-house produced Bst DNA Polymerase Large Fragment (5.46 ng/μL for LAMP and 7.31 ng/μL for RT-LAMP reactions).

Techniques: Diagnostic Assay, Fluorescence, Amplification, Staining, Incubation, DNA Amplification, Produced, RNA Amplification, Comparison, High Throughput Screening Assay, Control, Virus

Journal: medRxiv

Article Title: International Multi-site Implementation of Local Cell-Free Protein Biomanufacturing to Advance Health and Research Equity

doi: 10.1101/2025.07.25.25332228

Figure Lengend Snippet: (a) Countries where diagnostics were manufactured on-site and successfully used to establish disease diagnostic programs. Colors correspond to the data shown in subsequent panels, representing the countries where the data was collected. (b) The functionality of on-site-produced RT-LAMP reactions was further tested with RNA isolated from cultured SARS-CoV-2 virus. Fluorescence measurements after 20 minutes of incubation were plotted. Data are shown as mean ± SD, n = 3. (c) Patient trials for a locally produced SARS-CoV-2 diagnostic program were conducted in four different countries: Brazil, Colombia, Chile, and Canada. RNA samples isolated from patients were analyzed via RT-LAMP, where fluorescence increases indicate successful amplification. Fluorescence after 20 minutes (y-axis) was plotted against corresponding Ct values obtained via CDC RT-qPCR gold-standard assays (x-axis). In-house diagnostic tests demonstrated diagnostic accuracy ranging from 90% to 100%. The dashed line represents the threshold value defined for RT-qPCR analysis. See Supplementary Information for detailed analysis. (d) The functionality of on-site-produced RT-LAMP reactions was further tested with RNA isolated from cultured chikungunya virus. Fluorescence measurements after 30 minutes of incubation were plotted. Data are shown as mean ± SD, n = 3. (e) Patient trial for chikungunya virus was conducted in Brazil using locally produced, on-demand diagnostics. RNA samples isolated from patient samples were analyzed via RT- LAMP using 10 μM SYTO 9 Green fluorescent nucleic acid dye, where fluorescence increases indicated successful amplification. Fluorescence after 30 minutes (y-axis) was plotted against the corresponding Ct values obtained using the CDC RT-qPCR gold-standard assays (x-axis) in parallel. The dashed line represents the threshold value defined for RT-qPCR analysis . (f) Cultured Oropouche virus was processed using three methods: (1) commercial column extraction, (2) simple boiling at 95 °C for 2 minutes, and (3) direct use of the cultured virus without pretreatment. Each sample was tested using on-site-produced RT- LAMP reactions with LAMP fluorescent dye to detect the virus, with all three methods yielding successful detection. Data are shown as mean ± SD, n = 3. (g) Patient trial for Oropouche virus was conducted in Brazil using locally produced, on-demand diagnostics. RNA samples isolated from patient samples were analyzed via RT-LAMP using 1X LAMP fluorescent dye, where fluorescence increases indicated successful amplification. Fluorescence after 40 minutes (y-axis) was plotted against the corresponding Ct values obtained using the RT-qPCR gold-standard assays (x-axis). The dashed line represents the threshold value defined for RT-qPCR analysis. (h) In parallel with the CDC RT-qPCR gold standard, in-house diagnostic tests for SARS-CoV-2, chikungunya, and Oropouche viruses demonstrated accuracy ranging from 90% to 100%. During this project phase, RNA quality and integrity were verified in all patient samples using the human endogenous controls for Ribonuclease P (RT-qPCR) and Beta-actin (RT-LAMP). See Supplementary Information for detailed analysis (Tables S6-12). Abbreviations are: NTC, non-template control; CHIKV, chikungunya virus; OROV, Oropouche virus; Ct, cycle threshold.

Article Snippet: In brief, LAMP reaction mixtures contained 1X isothermal buffer (20 mM Tris-HCl, 10 mM (NH4)2SO4, 50 mM KCl, 2 mM MgSO4, 0.1% Tween 20, pH 8.8), 4 mM MgSO 4 , 1.4 mM deoxynucleotides triphosphates (dNTPs) (NEB, N0446S), 10X primer mix, and our in-house produced Bst DNA Polymerase Large Fragment (5.46 ng/μL for LAMP and 7.31 ng/μL for RT-LAMP reactions).

Techniques: Diagnostic Assay, Produced, Isolation, Cell Culture, Virus, Fluorescence, Incubation, Amplification, Quantitative RT-PCR, Extraction, Control

Journal: Life Science Alliance

Article Title: A lyophilized open-source RT-LAMP assay for molecular diagnostics in resource-limited settings

doi: 10.26508/lsa.202403167

Figure Lengend Snippet: (A) Comparison of DNA polymerase enzymes in RT-LAMP reactions. Detection rates of RT-LAMP reactions containing different DNA polymerases in addition to WarmStart RTx reverse transcriptase (NEB) and various concentrations of synthetic SARS-CoV-2 RNA. Results were obtained from four replicates per condition. (B) Comparison of reverse transcriptase enzymes in RT-LAMP reactions. Analogous to (A) but for different reverse transcriptase enzymes in combination with in-house Bst LF DNA polymerase. M-MuLV, Moloney murine leukemia virus reverse transcriptase; AMV, avian myeloblastosis virus reverse transcriptase; HIV-1, human immunodeficiency virus 1 reverse transcriptase. (C) Cross-contamination prevention in RT-LAMP via uracil-DNA glycosylase (UDG) enzymes. Diluted amounts of contaminating amplicons were added to RT-LAMP reactions containing different uracil-DNA glycosylase (UDG) enzymes and synthetic SARS-CoV-2 template. No UDG enzyme and nontemplate control reactions were included. Shown are time-to-threshold values from real-time fluorescence RT-LAMP reactions performed in duplicates. In-house BMTU UDG enzyme was tested against Antarctic Thermolabile UDG (NEB). (D) Thermostability test of BMTU UDG and commercial UDG enzymes. In-house BMTU UDG and commercial Antarctic Thermolabile UDG (NEB) were preincubated at different temperatures for 5 min. UDG enzyme was then added to qPCRs targeting either dTTP-containing DNA template or dUTP-containing DNA template. Cycle-to-threshold values (Ct) from duplicate reactions are shown for each condition. Crossed box = not determined. (E) Limit of detection of open-access and commercial RT-LAMP reactions. Reactions prepared from in-house Bst LF, HIV-1 RT, and BMTU UDG enzymes were compared with commercial reactions using 2X WarmStart LAMP Kit (NEB) containing engineered proprietary enzymes. Synthetic SARS-CoV-2 RNA at defined copy numbers was used as a template, and 20 replicates were performed per condition. Time-to-threshold values from real-time fluorescence RT-LAMP reactions are shown. (F) Specificity test of RT-LAMP reactions detecting different pathogens. RT-LAMP reactions were assembled with Bst LF and HIV-1 RT. Primers targeting SARS-CoV-2, Influenza A, or hRNase P were tested against SARS-CoV-2 RNA, Influenza A RNA (each individually spiked into HEK293-extracted RNA), HEK293-extracted RNA alone, and nuclease-free water as a no-template control. Reactions were performed in four replicates, and end-point relative fluorescent units are displayed. Source data are available for this figure.

Article Snippet: Further studies are required to assess long-term stability (e.g., 2 yr), as has been achieved by leading commercial products (e.g., LyoPrime WarmStart RT-LAMP Mix by New England Biolabs or TB LAMP by Eiken Chemical).

Techniques: Comparison, Reverse Transcription, Virus, Control, Fluorescence

Journal: Scientific reports

Article Title: A paper-based loop-mediated isothermal amplification assay for highly pathogenic avian influenza.

doi: 10.1038/s41598-025-95452-6

Figure Lengend Snippet: Fig. 1. Screening of LAMP primer sets for H5 subtype AIV by fluorescent LAMP assay using the NEB Warm Start DNA/RNA LAMP Kit.

Article Snippet: The master mix utilized for the assay was composed of 2.5 μl of 10 × HA LAMP primer mix, 12.5 μl of WarmStart® 2X Master Mix E1700 LAMP Kit (NEB, USA), and 5 μl 5 × LAMP fluorescent dye (diluted in nuclease-free water from 50X LAMP fluorescent dye provided with the kit) per reaction.

Techniques: Lamp Assay

Journal: Biosensors

Article Title: Magnetically Localized Detection of Amplified DNA Using Biotinylated and Fluorescent Primers and Magnetic Nanoparticles.

doi: 10.3390/bios15030195

Figure Lengend Snippet: Figure 2. Characterization of amplified DNA samples. (A) qPCR amplification kinetics. The SYBR Green fluorescent signal is plotted as a function of the cycle number. PCR: PCR positive control. Negative control: reaction without template DNA. Data are the average of three replicates. The standard deviation is smaller than the size of the symbols. The coefficient of variation of the specific signal is 1%. (B) LAMP amplification kinetics. The LAMP fluorescent dye signal is plotted as a function of time. LAMP: LAMP reaction with F3, B3, FIP, BIP, LF, and LB primers without modification. Negative control: same reaction without template DNA. FIP CY5: LAMP reaction with fluorescent FIP CY5 and biotinylated LF primer FIP CY5 LAMP kinetics. LF CY5: LF CY5 LAMP kinetics. No F3 B3: LAMP reaction without primers F3 and B3. No FIP BIP: LAMP reaction without primers FIP and BIP. No LF LB: LAMP reaction without primers LF and LB. Data are the average of three replicates. The coefficients of variation of the LAMP, FIP CY5, LF CY5, and No F3 B3 kinetics are 5%, 6%, 7%, and 13%, respectively. (C) Agarose gel (1%) electrophoresis analysis of the amplicons generated. Lane 1: Molecular weight marker (MWM). Lane 2: 461 bp PCR amplicon synthesized using biotinylated 5′-P Biotin and 3′-P cyanine 5. Lane 3: negative PCR control without template DNA. Lane 4: LAMP amplicons obtained with F3, B3, FIP, BIP, LF, and LB primers without modification. Lane 5: negative LAMP control without template DNA. Lane 6: FIP CY5 LAMP product. Lane 7: LF CY5 product. Lane 8: LAMP amplicons generated without primers F3 and B3. Lane 9: LAMP reaction without primers FIP and BIP. Lane 10: LAMP reaction without primers LF and LB. The gel shown is representative of at least 3 independent experiments.

Article Snippet: Quantitative LAMP reactions were performed in a 25 μL reaction mixture containing 1 μL of pUC19 plasmid DNA (1 ng), 12.5 μL of WarmStart® LAMP Kit (DNA and RNA), 0.5 μL of LAMP Fluorescent Dye, 8.5 μL of deionized water, and 2.5 μL LAMP primer mix, using a Bio-Rad CFX96 Real-Time System.

Techniques: Amplification, SYBR Green Assay, Positive Control, Negative Control, Standard Deviation, Modification, Agarose Gel Electrophoresis, Electrophoresis, Generated, Molecular Weight, Marker, Synthesized, Control

Journal: Biosensors

Article Title: Magnetically Localized Detection of Amplified DNA Using Biotinylated and Fluorescent Primers and Magnetic Nanoparticles.

doi: 10.3390/bios15030195

Figure Lengend Snippet: Figure 4. Magnetically localized fluorescent signals as a function of amplified DNA concentra- tion. One hundred sixty-eight or seven hundred fifty nanograms of biotinylated fluorescent DNA was generated by PCR (A,B) or LAMP using fluorescent FIP (C,D) or LF primers (E,F). Ampli- cons were captured on streptavidin-coated MNPs (blue circles in A,C,E; left picture in B,D,F) or ovalbumin-coated MNPs (black triangles in A) and analyzed with a MagIA analyzer. As controls, non-biotinylated PCR or LAMP products were generated and captured on streptavidin-coated MNPs (red squares in A,C,E; right picture in B,D,F). (A,C,E) Magnetically localized fluorescent (MLF) signals as a function of amplified DNA concentration. The MLF signal obtained in the absence of amplified DNA was subtracted from all measurements. The straight lines are a linear regression through the origin. The results are the average of 6 samples. (B,D,F) Representative cyanine 5 fluorescence images of biotinylated (left) or non-biontinylated (right) amplicons captured on micromagnets by streptavidin-coated MNPs. Images in B are acquired with 700 ms exposure time, and images in D and F are acquired with 50 ms exposure time.

Article Snippet: Quantitative LAMP reactions were performed in a 25 μL reaction mixture containing 1 μL of pUC19 plasmid DNA (1 ng), 12.5 μL of WarmStart® LAMP Kit (DNA and RNA), 0.5 μL of LAMP Fluorescent Dye, 8.5 μL of deionized water, and 2.5 μL LAMP primer mix, using a Bio-Rad CFX96 Real-Time System.

Techniques: Amplification, Generated, Concentration Assay, Fluorescence

Journal: Biosensors

Article Title: Magnetically Localized Detection of Amplified DNA Using Biotinylated and Fluorescent Primers and Magnetic Nanoparticles.

doi: 10.3390/bios15030195

Figure Lengend Snippet: Figure 5. Compared sensitivity of magnetically localized fluorescent technology and DNA intercala- tion for the detection of amplified DNA products. One nanogram of plasmid DNA was amplified by PCR (A) or by LAMP using an FIP CY5 (B) or LF CY5 (C) set of primers incorporating biotinylated and cyanine 5-modified primers in the presence of SYBR Green (PCR) or LAMP fluorescent dye (LAMP). The kinetics were monitored in parallel using magnetically localized fluorescent signals (MLF, circles) and DNA-intercalated fluorescent dyes (CFX, squares). The results are the average of 8 samples, except LAMP FIP CY5 (16 samples).

Article Snippet: Quantitative LAMP reactions were performed in a 25 μL reaction mixture containing 1 μL of pUC19 plasmid DNA (1 ng), 12.5 μL of WarmStart® LAMP Kit (DNA and RNA), 0.5 μL of LAMP Fluorescent Dye, 8.5 μL of deionized water, and 2.5 μL LAMP primer mix, using a Bio-Rad CFX96 Real-Time System.

Techniques: Amplification, Plasmid Preparation, Modification, SYBR Green Assay

Journal: Biosensors

Article Title: Magnetically Localized Detection of Amplified DNA Using Biotinylated and Fluorescent Primers and Magnetic Nanoparticles.

doi: 10.3390/bios15030195

Figure Lengend Snippet: Figure 6. Quantitative detection of template plasmid DNA. A total of 15 cycles of PCR (A) or 15 min of LAMP (B,C) were performed with the indicated amounts of template plasmid DNA in the presence of SYBR Green (A) or LAMP fluorescent dye (B,C) using biotinylated and cyanine-5-labeled primers. The samples were then processed to record MLF (circles) and CFX signals (squares). The results are the average of 8 samples.

Article Snippet: Quantitative LAMP reactions were performed in a 25 μL reaction mixture containing 1 μL of pUC19 plasmid DNA (1 ng), 12.5 μL of WarmStart® LAMP Kit (DNA and RNA), 0.5 μL of LAMP Fluorescent Dye, 8.5 μL of deionized water, and 2.5 μL LAMP primer mix, using a Bio-Rad CFX96 Real-Time System.

Techniques: Plasmid Preparation, SYBR Green Assay, Labeling