Journal: medRxiv

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

doi: 10.1101/2024.11.19.24317525

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, and 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 non-template 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 pre-incubated at different temperatures for 5 minutes. UDG enzyme was then added to qPCR reactions targeting either dTTP-containing DNA template or dUTP containing DNA template. Cycles 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 to commercial reactions using the 2X WarmStart LAMP Kit (NEB) containing engineered proprietary enzymes. Synthetic SARS-CoV-2 RNA at defined copy numbers was used as 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 hRNaseP were tested against SARS-CoV-2 RNA, Influenza A RNA (each individually spiked into HEK 293 extracted RNA), HEK 293 extracted RNA alone and nuclease-free water as no-template control. Reactions were performed in 4 replicates and end-point relative fluorescent units are displayed.

Article Snippet: To test different DNA polymerases, WarmStart® RTx Reverse Transcriptase (NEB) was added at 0.3 U/µl and Antarctic Thermolabile UDG (NEB) was added at 0.02 U/µl to all reactions.

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

Journal: medRxiv

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

doi: 10.1101/2024.11.19.24317525

Figure Lengend Snippet: (A) Time to threshold for DNA polymerase enzyme comparison in RT-LAMP. Time to threshold data for experiment shown in – A. Four replicates per condition were performed. (B) Time to threshold for reverse transcriptase enzyme comparison in RT-LAMP. Time to threshold data for experiment shown in – B. Four replicates per condition were performed. (C) Time to threshold for sensitivity test of RT-LAMP reactions with UDG enzymes. Reactions were prepared with Bst LF and HIV-1 RT. In-house BMTU UDG, Antarctic thermolabile UDG or no UDG enzymes were included. Four replicates per condition were performed. (D) Schematic representation for bead-LAMP workflow. Bead-LAMP was performed according to the protocol published previously . In brief, 100 µl of sample (TCEP/Betaine/ProteinaseK-inactivated sample) was mixed with 60 µl of magnetic bead slurry and left to incubate for 5 minutes to facilitate binding of nucleic acids to magnetic beads. The beads were separated on a magnet for 5 minutes or until the solution turned completely clear. Supernatant was discarded and 200 µl of 85% ethanol was added. After 30 seconds, the ethanol was removed, and beads were left to dry out in the tube for a period of 3 minutes. RT-LAMP reaction mix was added directly on top of the dried beads and beads were resuspended in the reaction mix. The reaction vessel was capped/sealed and incubated for 35 minutes at 63°C. (E) Bead-LAMP increases sensitivity of open-source RT-LAMP reactions. Samples were prepared by diluting synthetic SARS-CoV-2 RNA in inactivated negative gargle sample in a serial dilution. The samples were tested by RT-LAMP and bead-LAMP in parallel to demonstrate the sensitivity boost provided by the simple bead-enrichment protocol.

Article Snippet: To test different DNA polymerases, WarmStart® RTx Reverse Transcriptase (NEB) was added at 0.3 U/µl and Antarctic Thermolabile UDG (NEB) was added at 0.02 U/µl to all reactions.

Techniques: Comparison, Reverse Transcription, Binding Assay, Magnetic Beads, Incubation, Serial Dilution

Journal: medRxiv

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

doi: 10.1101/2024.11.19.24317525

Figure Lengend Snippet: (A) Schematic representation of lyophilization options for freeze-drying RT-LAMP enzymes and reagents. Glycerol free enzymes can be lyophilized either as a concentrated enzyme mix or as a reagent mixture with dNTPs and primers. Trehalose is used in both options as a cryoprotectant. Mixtures are flash-frozen in liquid nitrogen, lyophilized using a freeze-dryer and stored in dry conditions at various temperatures for transport. Before use, lyophilized mixtures are reconstituted. (B) Performance of reactions assembled with reconstituted RT-LAMP enzyme mix. RT-LAMP enzyme mix was lyophilized and stored at 22°C, 4°C and -20°C. After 30 days, lyophilized enzymes were reconstituted and used to assemble RT-LAMP reactions; control RT-LAMP reactions were prepared with non-lyophilized enzymes stored at -20°C. Reactions were tested on a four-fold, five-step dilution series of a positive sample in four replicates by RT-LAMP, recording the time-to-threshold for each reaction. In addition, the sample dilutions were measured by direct-input RT-qPCR, and Ct values are displayed under the columns. (C) Time to threshold for reactions prepared using a reconstituted RT-LAMP reagent mix. RT-LAMP reagent mix was lyophilized and stored at -20°C, 4°C, 22°C and 37°C. After 40 days, RT-LAMP reagents were reconstituted using the reconstitution buffer and reactions were tested on a four-fold, three-step dilution series of a positive sample in duplicates by RT-LAMP, recording the time-to-threshold for each reaction. In addition, the sample dilutions were measured by direct-input RT-qPCR, and Ct values are displayed under the columns. (D) Sensitivity comparison of RT-LAMP reactions assembled from lyophilized and non-lyophilized RT- LAMP reagents. Lyophilized and freshly prepared reactions were compared in 20 replicates using a dilution of SARS-CoV-2 synthetic RNA (Twist Biosciences) to assess the detection limit. Time to threshold values are shown. (E) Forced contamination experiment using lyophilized RT-LAMP reagents. In-house BMTU UDG enzyme, commercially available Antarctic Thermolabile UDG (New England Biolabs) and no UDG were included in three respective RT-LAMP reagent mixes for lyophilization. After freeze-drying, the reagent mixes were reconstituted into RT-LAMP master mixes and compared to freshly prepared reactions in a forced contamination experiment as described earlier ( – C). (F) HNB colorimetric readout is compatible with lyophilized RT-LAMP reagent mixture. Reactions were prepared in parallel from cold-stored enzymes (left) and lyophilized reaction mix (right) to compare colorimetric readout. Smartphone images were taken after lyophilization/preparation of master mix (top row), after addition of sample (middle row) and after a 35-minute incubation at 63°C (bottom row) to showcase the color change. (G) Proof-of-concept multi-pathogen respiratory virus RT-LAMP test. An 8-well PCR strip was filled with singe-reaction aliquots of RT-LAMP reagent mix for different target RNAs and freeze-dried. A multi-pathogen test strip targeting SARS-CoV-2, human coronavirus NL63 (HCoV-NL63), Influenza A, Respiratory Syncytial Virus A (RSV A) and human RNaseP (PC, positive control) was prepared from lyophilized reagents. Shown are the HNB RT-LAMP colorimetric results after reconstitution and addition of mock respiratory sample containing the respective pathogen RNA. A light blue color indicates a positive result.

Article Snippet: To test different DNA polymerases, WarmStart® RTx Reverse Transcriptase (NEB) was added at 0.3 U/µl and Antarctic Thermolabile UDG (NEB) was added at 0.02 U/µl to all reactions.

Techniques: Lyophilization, Control, Quantitative RT-PCR, Comparison, Incubation, Virus, Stripping Membranes, Positive Control