thermolabile proteinase k  (New England Biolabs)


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    Structured Review

    New England Biolabs thermolabile proteinase k
    Integration of TEC purification with USER barcoding. (A) Degradation of purified TECs using Thermolabile <t>Proteinase</t> K (TL Prot. K) followed by heat treatment at 65 °C. (B) Stepwise visualization of the USER barcoding procedure beginning from a single pool of purified TECs.
    Thermolabile Proteinase K, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 28 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/thermolabile proteinase k/product/New England Biolabs
    Average 94 stars, based on 28 article reviews
    Price from $9.99 to $1999.99
    thermolabile proteinase k - by Bioz Stars, 2022-09
    94/100 stars

    Images

    1) Product Images from "Preparation of E. coli RNA polymerase transcription elongation complexes for systematic RNA assays"

    Article Title: Preparation of E. coli RNA polymerase transcription elongation complexes for systematic RNA assays

    Journal: bioRxiv

    doi: 10.1101/2021.03.15.435517

    Integration of TEC purification with USER barcoding. (A) Degradation of purified TECs using Thermolabile Proteinase K (TL Prot. K) followed by heat treatment at 65 °C. (B) Stepwise visualization of the USER barcoding procedure beginning from a single pool of purified TECs.
    Figure Legend Snippet: Integration of TEC purification with USER barcoding. (A) Degradation of purified TECs using Thermolabile Proteinase K (TL Prot. K) followed by heat treatment at 65 °C. (B) Stepwise visualization of the USER barcoding procedure beginning from a single pool of purified TECs.

    Techniques Used: Purification

    2) Product Images from "Preparation of E. coli RNA polymerase transcription elongation complexes for systematic RNA assays"

    Article Title: Preparation of E. coli RNA polymerase transcription elongation complexes for systematic RNA assays

    Journal: bioRxiv

    doi: 10.1101/2021.03.15.435517

    Integration of TEC purification with USER barcoding. (A) Degradation of purified TECs using Thermolabile Proteinase K (TL Prot. K) followed by heat treatment at 65 °C. (B) Stepwise visualization of the USER barcoding procedure beginning from a single pool of purified TECs.
    Figure Legend Snippet: Integration of TEC purification with USER barcoding. (A) Degradation of purified TECs using Thermolabile Proteinase K (TL Prot. K) followed by heat treatment at 65 °C. (B) Stepwise visualization of the USER barcoding procedure beginning from a single pool of purified TECs.

    Techniques Used: Purification

    3) Product Images from "Novel method for multiplexed full-length single-molecule sequencing of the human mitochondrial genome"

    Article Title: Novel method for multiplexed full-length single-molecule sequencing of the human mitochondrial genome

    Journal: bioRxiv

    doi: 10.1101/2022.02.08.479581

    Cas9-mtDNA-enrichment, barcoding, pooling and demultiplexing approach for long-read sequencing. A schematic overview of the dual-guide targeting of full-length mtDNA with indicated gRNA-guided Cas9 cut sites. Samples can be targeted by several different gRNAs. Here we selected a pair of gRNAs on opposite sides of the circular mtDNA for each sample (1). After optional treatment of gDNA with Exonuclease V and dephosphorylation, samples are split into two aliquots for dual-guide targeted cleavage. Each cut site serves downstream as barcode in the analysis pipeline. The circular mtDNA molecules are opened, Cas9 is removed by Proteinase K digestion, which is followed by mtDNA dA-tailing and pooling of all of the aliquots. (2) ONT library preparation of the pooled samples and long-read sequencing on a nanopore flow cell. (3) The bioinformatics analysis encompasses basecalling, alignment, demultiplexing, variant calling and annotation.
    Figure Legend Snippet: Cas9-mtDNA-enrichment, barcoding, pooling and demultiplexing approach for long-read sequencing. A schematic overview of the dual-guide targeting of full-length mtDNA with indicated gRNA-guided Cas9 cut sites. Samples can be targeted by several different gRNAs. Here we selected a pair of gRNAs on opposite sides of the circular mtDNA for each sample (1). After optional treatment of gDNA with Exonuclease V and dephosphorylation, samples are split into two aliquots for dual-guide targeted cleavage. Each cut site serves downstream as barcode in the analysis pipeline. The circular mtDNA molecules are opened, Cas9 is removed by Proteinase K digestion, which is followed by mtDNA dA-tailing and pooling of all of the aliquots. (2) ONT library preparation of the pooled samples and long-read sequencing on a nanopore flow cell. (3) The bioinformatics analysis encompasses basecalling, alignment, demultiplexing, variant calling and annotation.

    Techniques Used: Sequencing, De-Phosphorylation Assay, Variant Assay

    4) Product Images from "Rapid and extraction-free detection of SARS-CoV-2 from saliva with colorimetric LAMP"

    Article Title: Rapid and extraction-free detection of SARS-CoV-2 from saliva with colorimetric LAMP

    Journal: medRxiv

    doi: 10.1101/2020.05.07.20093542

    Preliminary validation of RT-LAMP and qRT-PCR on clinical saliva samples A) Saliva samples from five COVID-19 positive individuals were subjected to the indicated pretreatment protocol followed by RT-LAMP. Heat = 55 °C for 15 minutes, 95 °C for 5 minutes, with or without proteinase K. Photographs for timepoints 10- and 30-minutes are shown. By 30 minutes, all positive controls were positive (yellow), and negative controls remained negative (magenta). 4 out of 5 samples pretreated with heat plus Proteinase K were called positive by colorimetric LAMP. B) qRT-PCR performed directly on crude pretreated saliva samples was qualitatively concordant with the LAMP assay. Samples 2-5 were positive (Ct
    Figure Legend Snippet: Preliminary validation of RT-LAMP and qRT-PCR on clinical saliva samples A) Saliva samples from five COVID-19 positive individuals were subjected to the indicated pretreatment protocol followed by RT-LAMP. Heat = 55 °C for 15 minutes, 95 °C for 5 minutes, with or without proteinase K. Photographs for timepoints 10- and 30-minutes are shown. By 30 minutes, all positive controls were positive (yellow), and negative controls remained negative (magenta). 4 out of 5 samples pretreated with heat plus Proteinase K were called positive by colorimetric LAMP. B) qRT-PCR performed directly on crude pretreated saliva samples was qualitatively concordant with the LAMP assay. Samples 2-5 were positive (Ct

    Techniques Used: Quantitative RT-PCR, Lamp Assay

    Final assay optimization, limit of detection analysis, and qRT-PCR A) Limit of detection analysis on optimized assay. qLAMP results from serial dilution of particles across three donor saliva samples, with or without proteinase K. ProK, proteinase K. Samples with Cts less than 50 (dotted red line) are called positive. Results are tallied in Table 1 . B) Paired box-plot of samples with less than 37.5 particles with or without proteinase K (ProK) shows that ProK reduces the mean and variability of Cts in these samples. C) Substituting TE for water in the fluorescent LAMP mastermix is compatible with samples that have been diluted in PBS. D) TE instead of water in the mastermix does not interfere with colorimetry. E) Increasing amounts of pretreated saliva containing three levels of virus were added to qLAMP reactions. Increasing amounts of pretreated saliva impede the LAMP reaction time. F) qRT-PCR on untreated and treated samples shows that heat with proteinase K treatment improves viral detection over untreated samples across all concentrations of virus. 1:1 TE samples are diluted but not heated.
    Figure Legend Snippet: Final assay optimization, limit of detection analysis, and qRT-PCR A) Limit of detection analysis on optimized assay. qLAMP results from serial dilution of particles across three donor saliva samples, with or without proteinase K. ProK, proteinase K. Samples with Cts less than 50 (dotted red line) are called positive. Results are tallied in Table 1 . B) Paired box-plot of samples with less than 37.5 particles with or without proteinase K (ProK) shows that ProK reduces the mean and variability of Cts in these samples. C) Substituting TE for water in the fluorescent LAMP mastermix is compatible with samples that have been diluted in PBS. D) TE instead of water in the mastermix does not interfere with colorimetry. E) Increasing amounts of pretreated saliva containing three levels of virus were added to qLAMP reactions. Increasing amounts of pretreated saliva impede the LAMP reaction time. F) qRT-PCR on untreated and treated samples shows that heat with proteinase K treatment improves viral detection over untreated samples across all concentrations of virus. 1:1 TE samples are diluted but not heated.

    Techniques Used: Quantitative RT-PCR, Serial Dilution, Colorimetric Assay

    Establishing isothermal pretreatment methods to improve compatibility with point-of-care testing A) Stabilizing buffers and ribonuclease inhibitor RNAsecure are compatible with LAMP. Colorimetric LAMP reactions proceed with saliva diluted 1:1 in either water, phosphate-buffered saline (PBS), TE (Tris 10 mM, EDTA 0.1 mM), and diluted TCEP (2.5 mM TCEP, 0.5 mM EDTA). B) A 15-minute isothermal (65 °C) heat pretreatment enables detection of 10 3 viral particles from saliva. Addition of RNAsecure improves sensitivity down to 10 2 viral particles. No additional benefit of diluted HUDSON reagents was seen compared to TE. C) Thermo-labile proteinase K treatment results in low sensitivity. While improved by RNAsecure, it performs worse than standard proteinase K with 95 °C inactivation, or mild heat treatments in (B). S = saliva control. D) Guanidium hydrochloride (40 mM) is compatible with the LAMP reaction. RNAsecure boosts the sensitivity in these conditions. Primer multiplexing in this buffer may further boost sensitivity to 10 1 particles per reaction. All reactions performed with NEB Gene N-A primers, except in (D) which also included Lamb et al . primers as indicated. E) Pulse centrifugation in a microfuge after pretreatment improves the reliability of LAMP detection. S, saliva.
    Figure Legend Snippet: Establishing isothermal pretreatment methods to improve compatibility with point-of-care testing A) Stabilizing buffers and ribonuclease inhibitor RNAsecure are compatible with LAMP. Colorimetric LAMP reactions proceed with saliva diluted 1:1 in either water, phosphate-buffered saline (PBS), TE (Tris 10 mM, EDTA 0.1 mM), and diluted TCEP (2.5 mM TCEP, 0.5 mM EDTA). B) A 15-minute isothermal (65 °C) heat pretreatment enables detection of 10 3 viral particles from saliva. Addition of RNAsecure improves sensitivity down to 10 2 viral particles. No additional benefit of diluted HUDSON reagents was seen compared to TE. C) Thermo-labile proteinase K treatment results in low sensitivity. While improved by RNAsecure, it performs worse than standard proteinase K with 95 °C inactivation, or mild heat treatments in (B). S = saliva control. D) Guanidium hydrochloride (40 mM) is compatible with the LAMP reaction. RNAsecure boosts the sensitivity in these conditions. Primer multiplexing in this buffer may further boost sensitivity to 10 1 particles per reaction. All reactions performed with NEB Gene N-A primers, except in (D) which also included Lamb et al . primers as indicated. E) Pulse centrifugation in a microfuge after pretreatment improves the reliability of LAMP detection. S, saliva.

    Techniques Used: Multiplexing, Centrifugation

    Dilution, heat, and Proteinase K treatments improve SARS-CoV-2 detection from saliva A) Dilution of particle-containing saliva into water improved LAMP detection by at least two orders of magnitude from undetectable to ~10 3 particles per reaction. Heat treatment and heat treatment plus proteinase K further increased LAMP sensitivity to ~10 2 viral genome equivalents per reaction. *Replicate 3 used Lamb et al . primers but gave nearly identical results to NEB Gene N-A primers. B) Heat treatment with or without proteinase K increased LAMP sensitivity from 10 6 to ~10 2 viral genome equivalents in undiluted saliva. C) Multiplexed primers improved LAMP sensitivity. LAMP reactions using NEB Gene N-A primers alone or in combination with Yu et al . or Lamb et al . primers are shown. S = negative control saliva. Viral particles per reaction are indicated. D) Saliva pretreatments significantly improved LAMP sensitivity. Heat treatment improved limit of detection ( p = 6e-6, t -test, two-tailed vs ‘No Treatment’). Proteinase K treatment further improved heat treatment (p = 0.002, t -test, two-tailed vs ‘Heat’). Multiplexed primers increased the frequency of detection at ~ 10 1 particles / reaction. N = NEB Gene N-A. E) qRT-PCR on crude saliva using the CDC N1 probe showed increased sensitivity with either heat or proteinase K treatment ( p
    Figure Legend Snippet: Dilution, heat, and Proteinase K treatments improve SARS-CoV-2 detection from saliva A) Dilution of particle-containing saliva into water improved LAMP detection by at least two orders of magnitude from undetectable to ~10 3 particles per reaction. Heat treatment and heat treatment plus proteinase K further increased LAMP sensitivity to ~10 2 viral genome equivalents per reaction. *Replicate 3 used Lamb et al . primers but gave nearly identical results to NEB Gene N-A primers. B) Heat treatment with or without proteinase K increased LAMP sensitivity from 10 6 to ~10 2 viral genome equivalents in undiluted saliva. C) Multiplexed primers improved LAMP sensitivity. LAMP reactions using NEB Gene N-A primers alone or in combination with Yu et al . or Lamb et al . primers are shown. S = negative control saliva. Viral particles per reaction are indicated. D) Saliva pretreatments significantly improved LAMP sensitivity. Heat treatment improved limit of detection ( p = 6e-6, t -test, two-tailed vs ‘No Treatment’). Proteinase K treatment further improved heat treatment (p = 0.002, t -test, two-tailed vs ‘Heat’). Multiplexed primers increased the frequency of detection at ~ 10 1 particles / reaction. N = NEB Gene N-A. E) qRT-PCR on crude saliva using the CDC N1 probe showed increased sensitivity with either heat or proteinase K treatment ( p

    Techniques Used: Negative Control, Two Tailed Test, Quantitative RT-PCR

    5) Product Images from "Preparation of E. coli RNA polymerase transcription elongation complexes for systematic RNA assays"

    Article Title: Preparation of E. coli RNA polymerase transcription elongation complexes for systematic RNA assays

    Journal: bioRxiv

    doi: 10.1101/2021.03.15.435517

    Integration of TEC purification with USER barcoding. (A) Degradation of purified TECs using Thermolabile Proteinase K (TL Prot. K) followed by heat treatment at 65 °C. (B) Stepwise visualization of the USER barcoding procedure beginning from a single pool of purified TECs.
    Figure Legend Snippet: Integration of TEC purification with USER barcoding. (A) Degradation of purified TECs using Thermolabile Proteinase K (TL Prot. K) followed by heat treatment at 65 °C. (B) Stepwise visualization of the USER barcoding procedure beginning from a single pool of purified TECs.

    Techniques Used: Purification

    6) Product Images from "EccDNAs are apoptotic products with high innate immunostimulatory activity"

    Article Title: EccDNAs are apoptotic products with high innate immunostimulatory activity

    Journal: Nature

    doi: 10.1038/s41586-021-04009-w

    The circularity of eccDNAs, but not the sequence, is critical for their immunostimulant activity a , Bar graphs showing the relative IL-6 and TNF-α mRNA levels. Data is shown as mean ± S EM of replicates (n=4 per group) of a representative experiment in three independent experiments. b-c , Bar graphs showing the relative IL-6 and TNF-α mRNA levels ( b ), and protein (ELISA) levels ( c ). Data is shown as mean ± SEM of replicates (n=4 per group) of a representative experiment in three independent experiments. d , Diagram of DNA transfection efficiency and stability assay. Step-1: 5 Phosphorothioate (purple “*”) end-protected synthetic linear DNA (PS-syn-linear) and its circular form with equal number of phosphorothioate bonds (PS-Syn-circular) were transfected to BMDCs in the same way as in Fig. 5 for either 1 or 12 hours. Step-2: cells were lysed in 100 μl lysis buffer and treated with Thermoliable Proteinase K to prepare total DNA. Step-3: 4 μl total DNA was used for qPCR with a set of primers that amplify a 117 bp fragment in both linear and circular DNA. e , qPCR analysis of the samples prepared as described in ( d ). Transfected DNA level was normalized to that of 10 ng/ml PS-syn-linear transfection (n= 4 or 6, as indicated by dotes). Bars indicate mean ± SEM. of three independent experiments. f , 12 hours after transfection of indicated DNA at 30 ng/ml, medium was collected for ELISA essay, Data is shown as mean ± SEM of replicates (n=4 per group) of a representative experiment in three independent experiments. Comparisons in a-c were performed on biological replicates by Ordinary one-way ANOVA with Tukey’s multiple comparison test; two-tailed unpaired t tests ( e-f ). *, p
    Figure Legend Snippet: The circularity of eccDNAs, but not the sequence, is critical for their immunostimulant activity a , Bar graphs showing the relative IL-6 and TNF-α mRNA levels. Data is shown as mean ± S EM of replicates (n=4 per group) of a representative experiment in three independent experiments. b-c , Bar graphs showing the relative IL-6 and TNF-α mRNA levels ( b ), and protein (ELISA) levels ( c ). Data is shown as mean ± SEM of replicates (n=4 per group) of a representative experiment in three independent experiments. d , Diagram of DNA transfection efficiency and stability assay. Step-1: 5 Phosphorothioate (purple “*”) end-protected synthetic linear DNA (PS-syn-linear) and its circular form with equal number of phosphorothioate bonds (PS-Syn-circular) were transfected to BMDCs in the same way as in Fig. 5 for either 1 or 12 hours. Step-2: cells were lysed in 100 μl lysis buffer and treated with Thermoliable Proteinase K to prepare total DNA. Step-3: 4 μl total DNA was used for qPCR with a set of primers that amplify a 117 bp fragment in both linear and circular DNA. e , qPCR analysis of the samples prepared as described in ( d ). Transfected DNA level was normalized to that of 10 ng/ml PS-syn-linear transfection (n= 4 or 6, as indicated by dotes). Bars indicate mean ± SEM. of three independent experiments. f , 12 hours after transfection of indicated DNA at 30 ng/ml, medium was collected for ELISA essay, Data is shown as mean ± SEM of replicates (n=4 per group) of a representative experiment in three independent experiments. Comparisons in a-c were performed on biological replicates by Ordinary one-way ANOVA with Tukey’s multiple comparison test; two-tailed unpaired t tests ( e-f ). *, p

    Techniques Used: Sequencing, Activity Assay, Enzyme-linked Immunosorbent Assay, Transfection, Stability Assay, Lysis, Real-time Polymerase Chain Reaction, Two Tailed Test

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    New England Biolabs thermolabile proteinase k
    Integration of TEC purification with USER barcoding. (A) Degradation of purified TECs using Thermolabile <t>Proteinase</t> K (TL Prot. K) followed by heat treatment at 65 °C. (B) Stepwise visualization of the USER barcoding procedure beginning from a single pool of purified TECs.
    Thermolabile Proteinase K, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/thermolabile proteinase k/product/New England Biolabs
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    thermolabile proteinase k - by Bioz Stars, 2022-09
    95/100 stars
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    Integration of TEC purification with USER barcoding. (A) Degradation of purified TECs using Thermolabile Proteinase K (TL Prot. K) followed by heat treatment at 65 °C. (B) Stepwise visualization of the USER barcoding procedure beginning from a single pool of purified TECs.

    Journal: bioRxiv

    Article Title: Preparation of E. coli RNA polymerase transcription elongation complexes for systematic RNA assays

    doi: 10.1101/2021.03.15.435517

    Figure Lengend Snippet: Integration of TEC purification with USER barcoding. (A) Degradation of purified TECs using Thermolabile Proteinase K (TL Prot. K) followed by heat treatment at 65 °C. (B) Stepwise visualization of the USER barcoding procedure beginning from a single pool of purified TECs.

    Article Snippet: 150 μl of the remaining sample was mixed with 6 μl (1 μl per sample volume) of Thermolabile Proteinase K (NEB), incubated at 37 °C for 30 minutes, and then at 65 °C for 20 minutes.

    Techniques: Purification

    Cas9-mtDNA-enrichment, barcoding, pooling and demultiplexing approach for long-read sequencing. A schematic overview of the dual-guide targeting of full-length mtDNA with indicated gRNA-guided Cas9 cut sites. Samples can be targeted by several different gRNAs. Here we selected a pair of gRNAs on opposite sides of the circular mtDNA for each sample (1). After optional treatment of gDNA with Exonuclease V and dephosphorylation, samples are split into two aliquots for dual-guide targeted cleavage. Each cut site serves downstream as barcode in the analysis pipeline. The circular mtDNA molecules are opened, Cas9 is removed by Proteinase K digestion, which is followed by mtDNA dA-tailing and pooling of all of the aliquots. (2) ONT library preparation of the pooled samples and long-read sequencing on a nanopore flow cell. (3) The bioinformatics analysis encompasses basecalling, alignment, demultiplexing, variant calling and annotation.

    Journal: bioRxiv

    Article Title: Novel method for multiplexed full-length single-molecule sequencing of the human mitochondrial genome

    doi: 10.1101/2022.02.08.479581

    Figure Lengend Snippet: Cas9-mtDNA-enrichment, barcoding, pooling and demultiplexing approach for long-read sequencing. A schematic overview of the dual-guide targeting of full-length mtDNA with indicated gRNA-guided Cas9 cut sites. Samples can be targeted by several different gRNAs. Here we selected a pair of gRNAs on opposite sides of the circular mtDNA for each sample (1). After optional treatment of gDNA with Exonuclease V and dephosphorylation, samples are split into two aliquots for dual-guide targeted cleavage. Each cut site serves downstream as barcode in the analysis pipeline. The circular mtDNA molecules are opened, Cas9 is removed by Proteinase K digestion, which is followed by mtDNA dA-tailing and pooling of all of the aliquots. (2) ONT library preparation of the pooled samples and long-read sequencing on a nanopore flow cell. (3) The bioinformatics analysis encompasses basecalling, alignment, demultiplexing, variant calling and annotation.

    Article Snippet: The mtDNA digestion was followed by incubation with 0.12 units of Thermolabile Proteinase K treatment (NEB) for 15 min at 37°C and 10 min at 55°C for inactivation.

    Techniques: Sequencing, De-Phosphorylation Assay, Variant Assay

    Preliminary validation of RT-LAMP and qRT-PCR on clinical saliva samples A) Saliva samples from five COVID-19 positive individuals were subjected to the indicated pretreatment protocol followed by RT-LAMP. Heat = 55 °C for 15 minutes, 95 °C for 5 minutes, with or without proteinase K. Photographs for timepoints 10- and 30-minutes are shown. By 30 minutes, all positive controls were positive (yellow), and negative controls remained negative (magenta). 4 out of 5 samples pretreated with heat plus Proteinase K were called positive by colorimetric LAMP. B) qRT-PCR performed directly on crude pretreated saliva samples was qualitatively concordant with the LAMP assay. Samples 2-5 were positive (Ct

    Journal: medRxiv

    Article Title: Rapid and extraction-free detection of SARS-CoV-2 from saliva with colorimetric LAMP

    doi: 10.1101/2020.05.07.20093542

    Figure Lengend Snippet: Preliminary validation of RT-LAMP and qRT-PCR on clinical saliva samples A) Saliva samples from five COVID-19 positive individuals were subjected to the indicated pretreatment protocol followed by RT-LAMP. Heat = 55 °C for 15 minutes, 95 °C for 5 minutes, with or without proteinase K. Photographs for timepoints 10- and 30-minutes are shown. By 30 minutes, all positive controls were positive (yellow), and negative controls remained negative (magenta). 4 out of 5 samples pretreated with heat plus Proteinase K were called positive by colorimetric LAMP. B) qRT-PCR performed directly on crude pretreated saliva samples was qualitatively concordant with the LAMP assay. Samples 2-5 were positive (Ct

    Article Snippet: We also tested thermolabile proteinase K from NEB (P8111S).

    Techniques: Quantitative RT-PCR, Lamp Assay

    Final assay optimization, limit of detection analysis, and qRT-PCR A) Limit of detection analysis on optimized assay. qLAMP results from serial dilution of particles across three donor saliva samples, with or without proteinase K. ProK, proteinase K. Samples with Cts less than 50 (dotted red line) are called positive. Results are tallied in Table 1 . B) Paired box-plot of samples with less than 37.5 particles with or without proteinase K (ProK) shows that ProK reduces the mean and variability of Cts in these samples. C) Substituting TE for water in the fluorescent LAMP mastermix is compatible with samples that have been diluted in PBS. D) TE instead of water in the mastermix does not interfere with colorimetry. E) Increasing amounts of pretreated saliva containing three levels of virus were added to qLAMP reactions. Increasing amounts of pretreated saliva impede the LAMP reaction time. F) qRT-PCR on untreated and treated samples shows that heat with proteinase K treatment improves viral detection over untreated samples across all concentrations of virus. 1:1 TE samples are diluted but not heated.

    Journal: medRxiv

    Article Title: Rapid and extraction-free detection of SARS-CoV-2 from saliva with colorimetric LAMP

    doi: 10.1101/2020.05.07.20093542

    Figure Lengend Snippet: Final assay optimization, limit of detection analysis, and qRT-PCR A) Limit of detection analysis on optimized assay. qLAMP results from serial dilution of particles across three donor saliva samples, with or without proteinase K. ProK, proteinase K. Samples with Cts less than 50 (dotted red line) are called positive. Results are tallied in Table 1 . B) Paired box-plot of samples with less than 37.5 particles with or without proteinase K (ProK) shows that ProK reduces the mean and variability of Cts in these samples. C) Substituting TE for water in the fluorescent LAMP mastermix is compatible with samples that have been diluted in PBS. D) TE instead of water in the mastermix does not interfere with colorimetry. E) Increasing amounts of pretreated saliva containing three levels of virus were added to qLAMP reactions. Increasing amounts of pretreated saliva impede the LAMP reaction time. F) qRT-PCR on untreated and treated samples shows that heat with proteinase K treatment improves viral detection over untreated samples across all concentrations of virus. 1:1 TE samples are diluted but not heated.

    Article Snippet: We also tested thermolabile proteinase K from NEB (P8111S).

    Techniques: Quantitative RT-PCR, Serial Dilution, Colorimetric Assay

    Establishing isothermal pretreatment methods to improve compatibility with point-of-care testing A) Stabilizing buffers and ribonuclease inhibitor RNAsecure are compatible with LAMP. Colorimetric LAMP reactions proceed with saliva diluted 1:1 in either water, phosphate-buffered saline (PBS), TE (Tris 10 mM, EDTA 0.1 mM), and diluted TCEP (2.5 mM TCEP, 0.5 mM EDTA). B) A 15-minute isothermal (65 °C) heat pretreatment enables detection of 10 3 viral particles from saliva. Addition of RNAsecure improves sensitivity down to 10 2 viral particles. No additional benefit of diluted HUDSON reagents was seen compared to TE. C) Thermo-labile proteinase K treatment results in low sensitivity. While improved by RNAsecure, it performs worse than standard proteinase K with 95 °C inactivation, or mild heat treatments in (B). S = saliva control. D) Guanidium hydrochloride (40 mM) is compatible with the LAMP reaction. RNAsecure boosts the sensitivity in these conditions. Primer multiplexing in this buffer may further boost sensitivity to 10 1 particles per reaction. All reactions performed with NEB Gene N-A primers, except in (D) which also included Lamb et al . primers as indicated. E) Pulse centrifugation in a microfuge after pretreatment improves the reliability of LAMP detection. S, saliva.

    Journal: medRxiv

    Article Title: Rapid and extraction-free detection of SARS-CoV-2 from saliva with colorimetric LAMP

    doi: 10.1101/2020.05.07.20093542

    Figure Lengend Snippet: Establishing isothermal pretreatment methods to improve compatibility with point-of-care testing A) Stabilizing buffers and ribonuclease inhibitor RNAsecure are compatible with LAMP. Colorimetric LAMP reactions proceed with saliva diluted 1:1 in either water, phosphate-buffered saline (PBS), TE (Tris 10 mM, EDTA 0.1 mM), and diluted TCEP (2.5 mM TCEP, 0.5 mM EDTA). B) A 15-minute isothermal (65 °C) heat pretreatment enables detection of 10 3 viral particles from saliva. Addition of RNAsecure improves sensitivity down to 10 2 viral particles. No additional benefit of diluted HUDSON reagents was seen compared to TE. C) Thermo-labile proteinase K treatment results in low sensitivity. While improved by RNAsecure, it performs worse than standard proteinase K with 95 °C inactivation, or mild heat treatments in (B). S = saliva control. D) Guanidium hydrochloride (40 mM) is compatible with the LAMP reaction. RNAsecure boosts the sensitivity in these conditions. Primer multiplexing in this buffer may further boost sensitivity to 10 1 particles per reaction. All reactions performed with NEB Gene N-A primers, except in (D) which also included Lamb et al . primers as indicated. E) Pulse centrifugation in a microfuge after pretreatment improves the reliability of LAMP detection. S, saliva.

    Article Snippet: We also tested thermolabile proteinase K from NEB (P8111S).

    Techniques: Multiplexing, Centrifugation

    Dilution, heat, and Proteinase K treatments improve SARS-CoV-2 detection from saliva A) Dilution of particle-containing saliva into water improved LAMP detection by at least two orders of magnitude from undetectable to ~10 3 particles per reaction. Heat treatment and heat treatment plus proteinase K further increased LAMP sensitivity to ~10 2 viral genome equivalents per reaction. *Replicate 3 used Lamb et al . primers but gave nearly identical results to NEB Gene N-A primers. B) Heat treatment with or without proteinase K increased LAMP sensitivity from 10 6 to ~10 2 viral genome equivalents in undiluted saliva. C) Multiplexed primers improved LAMP sensitivity. LAMP reactions using NEB Gene N-A primers alone or in combination with Yu et al . or Lamb et al . primers are shown. S = negative control saliva. Viral particles per reaction are indicated. D) Saliva pretreatments significantly improved LAMP sensitivity. Heat treatment improved limit of detection ( p = 6e-6, t -test, two-tailed vs ‘No Treatment’). Proteinase K treatment further improved heat treatment (p = 0.002, t -test, two-tailed vs ‘Heat’). Multiplexed primers increased the frequency of detection at ~ 10 1 particles / reaction. N = NEB Gene N-A. E) qRT-PCR on crude saliva using the CDC N1 probe showed increased sensitivity with either heat or proteinase K treatment ( p

    Journal: medRxiv

    Article Title: Rapid and extraction-free detection of SARS-CoV-2 from saliva with colorimetric LAMP

    doi: 10.1101/2020.05.07.20093542

    Figure Lengend Snippet: Dilution, heat, and Proteinase K treatments improve SARS-CoV-2 detection from saliva A) Dilution of particle-containing saliva into water improved LAMP detection by at least two orders of magnitude from undetectable to ~10 3 particles per reaction. Heat treatment and heat treatment plus proteinase K further increased LAMP sensitivity to ~10 2 viral genome equivalents per reaction. *Replicate 3 used Lamb et al . primers but gave nearly identical results to NEB Gene N-A primers. B) Heat treatment with or without proteinase K increased LAMP sensitivity from 10 6 to ~10 2 viral genome equivalents in undiluted saliva. C) Multiplexed primers improved LAMP sensitivity. LAMP reactions using NEB Gene N-A primers alone or in combination with Yu et al . or Lamb et al . primers are shown. S = negative control saliva. Viral particles per reaction are indicated. D) Saliva pretreatments significantly improved LAMP sensitivity. Heat treatment improved limit of detection ( p = 6e-6, t -test, two-tailed vs ‘No Treatment’). Proteinase K treatment further improved heat treatment (p = 0.002, t -test, two-tailed vs ‘Heat’). Multiplexed primers increased the frequency of detection at ~ 10 1 particles / reaction. N = NEB Gene N-A. E) qRT-PCR on crude saliva using the CDC N1 probe showed increased sensitivity with either heat or proteinase K treatment ( p

    Article Snippet: We also tested thermolabile proteinase K from NEB (P8111S).

    Techniques: Negative Control, Two Tailed Test, Quantitative RT-PCR