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## s12010-023-04799-x
- 标题：Characterization and Validation of a Lyophilized Loop-Mediated Isothermal Amplification Method for the Detection of Esox lucius.
- 期刊/年：Applied biochemistry and biotechnology 2024｜DOI: 10.1007/s12010-023-04799-x｜PMCID: PMC11401793｜通道: -
- 本地全文：/Users/liuxinyuan/Desktop/Hermes输出-工作类/知识沉淀/_全文/OA/10.1007_s12010-023-04799-x.txt

**摘要（直引节选）**：Keywords Loop-mediated isothermal amplification Lyophilization Freeze-dried beads Esox lucius Storage Characterization and validation Ministry of Education and Research Norway University Of South-Eastern Norway Open access funding provided by University Of South-Eastern Norway pmc-status-qastatus 0 pmc-status-live yes pmc-status-embargo no pmc-status-released yes pmc-prop-open-access yes pmc-prop-olf no pmc-prop-manuscript no pmc-prop-legally-suppressed no pmc-prop-has-pdf yes pmc-prop-has-supplement no pmc-prop-pdf-only no pmc-prop-suppress-copyright no pmc-prop-is-real-version no pmc-prop-is-scanned-article no pmc-prop-preprint no pmc-prop-in-epmc yes pmc-license-ref CC BY issue-copyright-statement © Springer Science+Business Media, LLC, part of Springer Nature 2024

**含数值句（直引）**：
- The lyophilized beads were stored at two different temperatures, at 20 °C and 4 °C, and tested for biological activity after different time intervals.
- The result shows that lyophilized beads are bioactive for almost 30 days when stored at 20 °C, while beads at 4 °C did not lose their bioactivity after storage for up to one year.
- Regarding the assessment of endangered species, the International Union for Conservation of Nature (IUCN) has expanded the global percentage of threatened species to 51% [ 7 ].
- The key feature of the LAMP technique is that, it amplifies nucleic acids under isothermal conditions in the range of 61–65 °C.
- Batches of lyophilized LAMP reagents with designed primers and different trehalose concentrations were prepared, characterized, and stored at 4 °C and 20 °C.
- The master mixture is expected to contain 10 mM dNTPs mix, 8 U Bst DNA polymerase, 20 mM Tris HCl, 50 mM KCl, 0.1 % Tween, 10 mM (NH 4 ) 2 SO 4 , 0.8 M betaine, and 8 mM MgSO 4 , and fluorescent dye could be FAM/SYBr Green for one reaction of amplification.
- Different concentration of trehalose includes 10 %, 15 %, and 20 % which was incorporated in the LAMP reaction mixture (w/v), containing optimized concentration of primers, enzyme, and cofactor ions that were subjected to the freeze-drying process.
- The reaction was carried out in 25 μL volume, as suggested by the warm start manual.
- The prepared LAMP reaction mixture was taken in an electronic pipette (eVol XR ) capable of dispensing 5 μL volume bead in each turn.
- LAMP Lyophilized Bead Validation The prepared beads were stored at two different temperature conditions, one at room temperature 20 °C and another at 4 °C in a refrigerator.
- Then, five beads were rehydrated with 24 μL of RNase-free water and tested towards Esox lucius DNA template (1 μL) at different time periods;
- The reaction was carried out at 65 °C for 1 h and set with 80 cycles.
- The amplicons were also quantified by fluorometer (Invitrogen, Qubit 4 Fluorometer) and viewed in 1 % agarose gel electrophoresis with ladder.
- Three trehalose concentrations, 10 %, 15 %, and 20 % (w/v), were used in the LAMP mixture beads (Fig.
- Several batches of 5 μL lyophilized beads were stored over time, in separate sealed glass ampoules both at room temperature 20 °C and at 4 °C in a refrigerator.
- 1 The LAMP mixture beads in different concentrations of trehalose ( a ) 10 %, ( b ) 15 %, and ( c ) 20 % weight percent Surface Dimensions and Morphology A scanning electron microscope was used to observe the objects in the micron to nano level range.
- The physical shape of the 5 μL beads was found to be round to oval shape, depending on the needle tip and dispensing time.
- The dimension of the lyophilized beads was found to be in the range of 1.1 to 1.8 mm among the three trehalose concentration beads (Fig.
- It was noticed that a trehalose concentration of 10 % in the LAMP mixture beads readily absorbs moisture and shrinks immediately, while the increasing concentration of 15 % and 20 % was more stable when exposed to atmospheric air.
- In this context, the shrinkage pattern of the surface is seen for the 10 % trehalose-LAMP mixture bead, and a dense structural surface with a porous nature is visible for both the 15 % trehalose-LAMP mixture bead and 20 % trehalose-LAMP mixture bead.
- 2 ( i ) The dimensions of the 5 μL lyophilized beads (a) 10 % trehalose- LAMP mixture bead, (b) 15 % trehalose-LAMP mixture beads, and (c) 20 % trehalose-LAMP mixture beads.
- 3 The Raman spectra for ( a ) pure trehalose bead, ( b ) the 15 % trehalose-LAMP mixture bead, and ( c ) the 20 % trehalose-LAMP mixture bead Stability Study of Lyophilized Bead The trehalose-LAMP mixture beads were stored at room temperature to evaluate the activity of lyophilized reagents at different time intervals and the average Ct values (cycle threshold) of the amplification is indicated (Fig.
- The average Ct value for 10 % trehalose-LAMP mixture bead is 50.3, 42.4, 50.5, 45.5, and 55.4 for storage after the first day, third day, fifth day, seventh day and one month, respectively.
- After storage of one month, the average Ct for 15 % trehalose-LAMP mixture bead is slightly less than 10 % trehalose-LAMP mixture beads and much less than 20 % trehalose-LAMP mixture beads.
- The 95% confidence interval for the population mean of the 15 % trehalose-LAMP mixture beads was considerably less in one month storage, compared to the other two compositions of beads as shown in Table 2 (a), (b), and (c).
- Only the 15 % trehalose-LAMP mixture beads had a significantly lower Ct, when stored at room temperature for one month.

**工艺/辅料句（直引）**：
- Therefore, this work aims to lyophilize the entire reaction mixture as a single microbead, with enzyme, and LAMP primers towards the detection of mitochondrial cytochrome B (Cyt B), a housekeeping gene in Esox lucius .
- Analytical and molecular techniques were performed to characterize and validate the lyophilized beads, respectively.
- Our study therefore focuses on constructing all necessary reaction components in a freeze-dried bead form, which is well suited for transport and storage.
- Such freeze-dried reagent beads can be easily placed inside a reaction chamber of lab-on-a-chip platform.
- Lyophilization is the best process for preserving biomolecules such as proteins, enzymes, and pharmaceutical liposomes, including a disaccharide as a key protective agent.
- The freeze-drying process consists of two main steps, namely freezing and drying under vacuum.
- The drying process itself is further classified into (i) the primary drying where frozen water is removed and (ii) the secondary drying where unfrozen (bound) water is removed [ 18 ].
- Commonly, named disaccharides such as sucrose and trehalose are used as cryoprotectants [ 19 , 20 ].
- In this study, we used trehalose which is a good glass former that helps to stabilize the biomolecules during and after the freeze-drying.
- The important glass transition factor of disaccharides is high in trehalose and acts as a support under higher temperature storage condition.
- This has been shown in previous studies by experimenting the stability of DNA modifying enzyme with 0.3 M trehalose compared to other saccharides such as sucrose, sorbitol, mannitol, and galactitol [ 21 ].
- Studies have revealed that DNA damage will be significantly reduced during the freeze-drying process of biomolecules, although DNA damage could not be avoided during room temperature storage.

## s41598-022-11144-5
- 标题：A lyophilized colorimetric RT-LAMP test kit for rapid, low-cost, at-home molecular testing of SARS-CoV-2 and other pathogens.
- 期刊/年：Scientific reports 2022｜DOI: 10.1038/s41598-022-11144-5｜PMCID: PMC9052177｜通道: -
- 本地全文：/Users/liuxinyuan/Desktop/Hermes输出-工作类/知识沉淀/_全文/OA/10.1038_s41598-022-11144-5.txt

**摘要（直引节选）**： pmc Sci Rep Sci Rep 1579 scirep 101563288 Scientific Reports 2045-2322 Nature Publishing Group PMC9052177 PMC9052177.2 9052177 9054813 35487969 10.1038/s41598-022-11144-5 11144 2 Article A lyophilized colorimetric RT-LAMP test kit for rapid, low-cost, at-home molecular testing of SARS-CoV-2 and other pathogens Song Xin xin.song@duke.edu 1 2 3 Coulter Felicity J. 4 Yang Ming 3 Smith Jessica L. 5 Tafesse Fikadu G. 4 Messer William B. messer@ohsu.edu 4 6 7 Reif John H. reif@cs.duke.edu 1 3 1 grid.26009.3d 0000 0004 1936 7961 Department of Electrical and Computer Engineering, Duke University, Durham, NC 27708 USA 2 grid.26009.3d 0000 0004 1936 7961 Department of Biomedical Engineering, Duke University, Durham, NC 27708 USA 3 grid.26009.3d 0000 0004 1936 7961 Department of Computer Science, Duke University, Durham, NC 27708 USA 4 grid.5288.7 0000 0000 9758 5690 Department of Molecular Microbiology and Immunology, Oregon Health and Science University, Portland, OR 97239 USA 5 grid.5288.7 0000 0000 9758 5690 Vaccine and Gene Therapy Institute, Oregon Health and Science University, Beaverton, OR 97006 USA 6 grid.5288.7 0000 0000 9758 5690 Department of Medicine, Division of Infectious Dis

**含数值句（直引）**：
- pmc Sci Rep Sci Rep 1579 scirep 101563288 Scientific Reports 2045-2322 Nature Publishing Group PMC9052177 PMC9052177.2 9052177 9054813 35487969 10.1038/s41598-022-11144-5 11144 2 Article A lyophilized colorimetric RT-LAMP test kit for rapid, low-cost, at-home molecular testing of SARS-CoV-2 and other pathogens Song Xin xin.song@duke.edu 1 2 3 Coulter Felicity J.
- The entire sample-to-answer workflow takes < 60 min, including noninvasive sample collection, one-step RNA preparation, reverse-transcription loop-mediated isothermal amplification (RT-LAMP) in a thermos, and direct visual inspection of a colorimetric test result.
- 1 ) takes < 60 min, including noninvasive sample collection (anterior nasal swab or alternatively gingival swab), quick extraction-free RNA preparation, optimized RT-LAMP reaction in a thermos, and finally a colorimetric interpretation of the test result.
- Step 3: Add 95 °C hot water into a thermos.
- Ensuring the sample collection tube lid is tightly secured, place inside the thermos and close the lid, to incubate for 10 min.
- Step 4: Take out the collection tube and cool it on ice for 5 min, allowing any debris to settle to the bottom.
- Use a disposable transfer pipette to draw 20 μL of sample from the collection tube.
- Step 6: Use a thermometer or a temperature sticker (included in the kit) to adjust the water temperature to 65 °C in the thermos.
- RT-LAMP reactions rely on active enzymatic components (i.e., DNA polymerase and reverse transcriptase) that must be stored at a low temperature (typically − 20 °C).
- Instead, our simplified protocol enables one-pot lyophilization of all reagents needed for the colorimetric RT-LAMP in a single microtube (Table 1 ), and the entire lyophilization process can be completed in under 2 h (see Methods).
- Component Amount used per kit WarmStart ® colorimetric LAMP 2X master mix 10 µL 10X primer mix 2 µL 3 M Trehalose 3 µL 1 M GuHCl 0.8 µL Table 2 SARS-CoV-2 RT-LAMP primer set used in the optimized RT-LAMP test.
- S7 , our lyophilized assay (“3 M trehalose + 1 M GuHCl) lyo”) performed robustly across the entire temperature gradient tested (60.7–70.0 °C) with clear readout of true positives as early as 20 min and no false positives by 50 min of incubation at most temperatures within the temperature gradient.
- Assay validation According to in-house validation using synthesized SARS-CoV-2 RNA (Twist Bioscience), our test kit remains stable for at least 30 days at typical home-refrigeration temperature (4 °C) (Fig.
- S11 ) and 10 days at room temperature (~ 20 to 22 °C) (Fig.
- S14 ), achieving ≥ 95% analytical sensitivity and > 99% specificity with a reproducible limit of detection (LoD) down to 100 RNA copies per reaction (i.e., 5 copies/μL) under both storage conditions.
- Specificity remained at ≥ 99% for the simulated samples (Fig.
- Xuan Qin, OHSU Department of Pathology), finding that the RT-LAMP assay would be expected to have detected SARS-CoV-2 in approximately 4254 patients, the number of patients with a CT of < 26, yielding a real-world sensitivity of 72%.
- Three independent replicates of four-reactions per dilution were performed using samples from 227 copies per reaction to 17,473 copies per reaction.
- Red-dotted lines and grey area indicates 95% confidence interval around the dose–response curve.
- Straight dotted lines show 50 and 95% analytic sensitivity intercepts as predicted by the fitted curve.
- 95% limit of detection was 15,514 viral copies, 50% limit of detection was 1421 viral copies per reaction.
- bottom panels) versus 15,518 viral copies of heat-inactivated SARS-CoV-2 (top panels) in uninfected pooled anterior nasal swab media, demonstrating ≥ 99% analytical specificity and ≥ 95% analytical sensitivity at 15,518 copies/reaction.
- 4.86 × 10^4), B.1.1.7 (1.51 × 10^5), and B.1.351 (4.79 × 10^5).
- Figure 4 Validation of the RT-LAMP home test on additional heat inactivated SARS-CoV-2 variants (calculated viral copy numbers per tube): P.2 (6.60 × 10^4), B.1.2 (2.69 × 10^5), B.1.427 (5.05 × 10^5), B.1.429 (4.12 × 10^4), B.1.526 (2.72 × 10^5), B.1.617.1 (1.65 × 10^5) and B.1.617.2 (4.05 × 10^4).
- In addition to its low cost and simplicity, the test kit features a user-friendly home testing workflow that can be easily completed in under 1 h with no specialized instrumentation or trained personnel (Table 3 ).
- Notably, our test conveniently tolerates larger sample input volumes (i.e., as opposed to 1 μL to 5 μL sample volume commonly used in standard RT-LAMP assays, our test directly accepts 20 μL swab media sample to rehydrate the lyophilized reagents for a 20 μL RT-LAMP reaction).

**工艺/辅料句（直引）**：
- To facilitate long-term storage without cold-chain, a fast one-pot lyophilization protocol was developed to preserve all required biochemical reagents of the colorimetric RT-LAMP test in a single microtube.
- Notably, the lyophilized RT-LAMP assay demonstrated reduced false positives as well as enhanced tolerance to a wider range of incubation temperatures compared to solution-based RT-LAMP reactions.
- With a simple change of the primer set, our lyophilized RT-LAMP home test can be easily adapted as a low-cost surveillance platform for other pathogens and infectious diseases of global public health importance.
- To enable truly inexpensive, rapid, reliable at-home testing of COVID-19, we developed a simple all-in-one molecular home test kit based on lyophilized colorimetric RT-LAMP, requiring only a regular thermos and a thermometer to conduct the self-administered test.
- A rapid, one-pot lyophilization protocol was developed to quickly preserve all reagents needed for the colorimetric RT-LAMP test in a single microtube, facilitating long-term stability, inexpensive distribution, and convenient use of the home test kit.
- Step 5: Quickly dispense the sample into the lyophilized RT-LAMP reaction tube.
- To preserve the RT-LAMP reagents for home test use, we employed lyophilization, also known as freeze-drying, to extend the shelf-life of the test kit and facilitate simple test kit distribution, handling, and storage under convenient temperatures (e.g., at typical home-refrigeration temperature or at room temperature).
- A lyophilized test kit also reduces the number of pipetting steps to improve usability and minimize contamination 26 .
- However, lyophilization is typically an expensive and time-consuming process involving three stages including freezing, primary drying, and secondary drying which can be difficult to design and optimize 27 .
- In this work, we developed a fast, one-pot lyophilization process that minimizes the drying time by completing both the primary and secondary drying under a single condition 28 .
- Unlike prior lyophilization protocols developed for molecular biology assays, our protocol eliminates the need to separately lyophilize the reaction buffer and the enzymes 17 , 26 , 29 .
- We tested trehalose 30 – 32 and dextran 33 , 34 as candidate excipients to provide cryo- and lyoprotection during lyophilization, as well as enhanced stability for long-term storage.

## s41598-024-61163-7
- 标题：Development and evaluation of a lyophilization protocol for colorimetric RT-LAMP diagnostic assay for COVID-19.
- 期刊/年：Scientific reports 2024｜DOI: 10.1038/s41598-024-61163-7｜PMCID: PMC11078981｜通道: -
- 本地全文：/Users/liuxinyuan/Desktop/Hermes输出-工作类/知识沉淀/_全文/OA/10.1038_s41598-024-61163-7.txt

**摘要（直引节选）**：Molecular diagnostics involving nucleic acids (DNA and RNA) are regarded as extremely functional tools. During the 2020 global health crisis, efforts intensified to optimize the production and delivery of molecular diagnostic kits for detecting SARS-CoV-2. During this period, RT-LAMP emerged as a significant focus. However, the thermolability of the reagents used in this technique necessitates special low-temperature infrastructure for transport, storage, and conservation. These requirements limit distribution capacity and necessitate cost-increasing adaptations. Consequently, this report details the development of a lyophilization protocol for reagents in a colorimetric RT-LAMP diagnostic kit to detect SARS-CoV-2, facilitating room-temperature transport and storage. We conducted tests to identify the ideal excipients that maintain the molecular integrity of the reagents and ensure their stability during room-temperature storage and transport. The optimal condition identified involved adding 5% PEG 8000 and 75 mM trehalose to the RT-LAMP reaction, which enabled stability at room temperature for up to 28 days and yielded an analytical and diagnostic sensitivity and specificity of 83.33% and 90%, respectively, for detecting SARS-CoV-2. This study presents the results of a lyophilized colorimetric RT-LAMP COVID-19 detection assay with diagnostic sensitivity and specificity comparable to RT-qPCR, particularly in samples with high viral load.

**含数值句（直引）**：
- The optimal condition identified involved adding 5% PEG 8000 and 75 mM trehalose to the RT-LAMP reaction, which enabled stability at room temperature for up to 28 days and yielded an analytical and diagnostic sensitivity and specificity of 83.33% and 90%, respectively, for detecting SARS-CoV-2.
- Lyophilized samples with different compositions were compared to each other and to standard fresh reactions, which means SARS-CoV-2 RT-LAMP without any lyophilization process and submitted to 60 °C for 30 min along with lyophilized samples, containing 10 6 copies of SARS-CoV-2 RNA and SARS-CoV-2 negative RNA control.
- The initial substance analyzed was trehalose, tested at four concentrations ranging from 50 to 150 mM in 10 6 copies of SARS-CoV-2 RNA, SARS-CoV-2 negative RNA, and non-template control (NTC).
- We observed that trehalose at 75 mM yielded the best result.
- The second substance, arginine, was tested at eight concentrations ranging from 2.5 mM to 75 mM, with 10 mM showing the best performance.
- Following this initial observation, a combination of trehalose and arginine at 75 mM and 10 mM, respectively, was tested;
- Following the experimental design, Polyethylene Glycol (PEG) 2,000 and PEG 8,000 were lyophilized in different concentrations in combination with 75 mM trehalose and 10 mM arginine.
- Initially, PEG 2,000 was tested at four concentrations (ranging from 1 to 10%), with the 10% concentration demonstrating the best result when combined with both arginine and trehalose, compared to fresh controls.
- For PEG 8,000, the same tests were conducted, and the 5% concentration was identified as the most suitable.
- Subsequently, we prepared a combination of 10% PEG 2,000 and 5% PEG 8,000 in fresh control reactions for both SARS-CoV-2 positive and negative samples and observed no impact on amplification when assessing the final reaction color, indicating that PEG does not interfere with RT-LAMP.
- The last tested substance was Polyvinylpyrrolidone (PVP) 40,000, tested at 2% and 5% final concentrations in conjunction with 75 mM trehalose and 10 mM arginine.
- Condition Arginine Trehalose PEG 2,000 PEG 8,000 1 10 mM – 10% – 2 10 mM – – 5% 3 – 75 mM 10% – 4 – 75 mM – 5% Another phase of lyophilization standardization and optimization involved a visual analysis of the appearance of the "cake," the term used to describe the lyophilized product.
- Figure 1 below displays lyophilized samples with 10% PEG 2,000 and 5% PEG 8,000 combined with 75 mM trehalose and 10 mM arginine.
- Lyophilization time and color measurement After selecting four lyophilization conditions, the protocol focused on optimizing lyophilization time by comparing durations of five hours and overnight (1five hours and 30 min) to determine the best lyophilization time for the selected formulations.
- In each selected formulation from Table 1 , the analysis involved, after lyophilization and resuspension, adding 10 6 copies of SARS-CoV-2 RNA, SARS-CoV-2 negative RNA, and NTC.
- After amplification, the samples were analyzed in a spectrophotometer at 434 and 560 nm to obtain quantitative data complementary to the visual analyses.
- Figure 2 A and B—Result of spectrophotometry for four conditions and two lyophilization times, indicating the difference between absorbance by spectrophotometry at wavelengths of 434 nm and 560 nm (ΔDO), respectively, for positive SARS-CoV-2 samples, negative RNA and NTC.
- The conditions selected to continue the experiments were 10 mM arginine + 10% PEG 2000 and 75 mM trehalose + 5% PEG 8,000.
- We conducted three independent trials with 10 5 , 10 4 , 10 3 , and 10 2 copies of SARS-CoV-2 RNA, each lasting five hours.
- The results indicated that all reactions containing 10 3 copies of SARS-CoV-2 RNA tested positive under both lyophilization formulations.
- Specifically, the 75 mM trehalose + 5% PEG 8,000 condition resulted in a slightly orange-yellowish hue, indicative of a positive result yet clearly distinct from a negative outcome.
- Conversely, in the arginine-based formulation, amplification was observed up to 10 2 copies of SARS-CoV-2 RNA in 50% of the reactions, with negative reactions exhibiting an intermediate orange coloration.
- In comparison, all reactions at the same RNA concentration using the trehalose 75 mM + 5% PEG 8,000 formulation tested negative (Fig.
- Consequently, the approximate limit of detection (LoD) appears to be lower in the condition with 10 mM arginine + 10% PEG 2,000, ranging between 10 3 and 10 2 copies of SARS-CoV-2 RNA.
- Figure 3 Representative image of 10% PEG 2,000 + 10 mM arginine and 5% PEG 8,000 + 75 mM trehalose colorimetric SARS-CoV-2 RT-LAMP lyophilized conditions with 10 4 , 10 3 , and 10 2 SARS-CoV-2 RNA copies, demonstrating that with the first formulation, the color change occurs in the samples containing 10 2 copies, while in the second formulation, amplification occurs only up to 10 3 copies.
- We observed that all 20 clinical samples with Ct values lower than 29 for SARS-CoV-2, as detected by RT-qPCR in the condition with 10 mM arginine + 10% PEG 2,000, demonstrated amplification with corresponding color change.

**工艺/辅料句（直引）**：
- Consequently, this report details the development of a lyophilization protocol for reagents in a colorimetric RT-LAMP diagnostic kit to detect SARS-CoV-2, facilitating room-temperature transport and storage.
- We conducted tests to identify the ideal excipients that maintain the molecular integrity of the reagents and ensure their stability during room-temperature storage and transport.
- This study presents the results of a lyophilized colorimetric RT-LAMP COVID-19 detection assay with diagnostic sensitivity and specificity comparable to RT-qPCR, particularly in samples with high viral load.
- Keywords: Lyophilization, RT-LAMP, Colorimetric kit, COVID-19 Subject terms: Biochemistry, Biological techniques, Molecular biology status released display-pdf yes is-olf no is-manuscript no is-preprint no is-journal-matter no is-scanned no is-retracted no Received 2024 Feb 21;
- The lyophilization process, a preservation method used in amplification reactions to make the mixtures resistant to environmental conditions, addresses this issue.
- The advantages of the lyophilized product include storage at room temperature, increased product durability, ease of transportation (since it does not require temperature control), and conservation of material characteristics 7 .
- An optimized lyophilization protocol requires extensive and complex screening of various reagents at different concentrations and formulations, alongside numerous other variables such as equipment and the time and temperature of freezing prior to lyophilization.
- This manuscript introduces a novel approach by utilizing several lyoprotectants, different lyophilization properties, and performance evaluation in SARS-CoV-2 clinical samples.
- It presents the results of the development, optimization, and validation of the RT-LAMP reaction lyophilization process for detecting SARS-CoV-2.
- Results Lyophilization formulations evaluation As part of the initial protocol screening for the lyophilization of the colorimetric SARS-CoV-2 RT-LAMP reaction, five protective reagents were tested in various combinations.
- This initial protocol was conducted using a standard lyophilization process, as described in the Methods section.
- Table 1 Four compositions of lyophilization for colorimetric SARS-CoV-2 RT-LAMP using arginine, trehalose, PEG 2000, and PEG 8000.

## sj.embor.7400200
- 标题：Helicase-dependent isothermal DNA amplification.
- 期刊/年：EMBO reports 2004｜DOI: 10.1038/sj.embor.7400200｜PMCID: PMC1249482｜通道: -
- 本地全文：/Users/liuxinyuan/Desktop/Hermes输出-工作类/知识沉淀/_全文/OA/10.1038_sj.embor.7400200.txt

**摘要（直引节选）**：Polymerase chain reaction is the most widely used method for in vitro DNA amplification. However, it requires thermocycling to separate two DNA strands. In vivo , DNA is replicated by DNA polymerases with various accessory proteins, including a DNA helicase that acts to separate duplex DNA. We have devised a new in vitro isothermal DNA amplification method by mimicking this in vivo mechanism. Helicase-dependent amplification (HDA) utilizes a DNA helicase to generate single-stranded templates for primer hybridization and subsequent primer extension by a DNA polymerase. HDA does not require thermocycling. In addition, it offers several advantages over other isothermal DNA amplification methods by having a simple reaction scheme and being a true isothermal reaction that can be performed at one temperature for the entire process. These properties offer a great potential for the development of simple portable DNA diagnostic devices to be used in the field and at the point-of-care.

**含数值句（直引）**：
- Copyright © 2004, European Molecular Biology Organization PMC Copyright notice PMCID: PMC1249482 PMID: 15247927 Abstract Polymerase chain reaction is the most widely used method for in vitro DNA amplification.
- After a 1 hr incubation period at 37°C, a 110-bp amplification product was observed on a 2% agarose gel ( Fig 2 , lane 1).
- A two-step HDA reaction, with a 1 h incubation at 37°C, was performed in the presence of all components (lane 1) including a pUC19-derived plasmid DNA (0.035 pmol), primer-1224 (10 pmol) and primer-1233 (10 pmol), UvrD helicase (100 ng), MutL (400 ng), T4 gene 32 protein (4.5 μg), ATP (0.15 μmol) and exo − Klenow polymerase (5 U).
- In the absence of T4 gene 32 protein, again no amplification product was observed ( Fig 2A , lane 4), indicating that SSB is required in this reaction, probably to prevent reassociation of the complementary ssDNA templates at 37°C.
- Target sequences up to 400 bp can be efficiently amplified from plasmid DNA, beyond which the yield drops markedly (data not shown).
- The amount of template was varied from 10 7 to 10 3 copies of the T.
- With 10 3 copies of initial target, about 10 ng of products were generated, which corresponds to 10 10 molecules of the 123-bp fragment.
- A two-step HDA reaction, with a 3 h incubation at 37°C, was performed in the presence of primer Ea136for (20 pmol) and primer Ea136rev (20 pmol), UvrD helicase (100 ng), MutL (800 ng), T4 gene 32 protein (4.5 μg), ATP (0.15 μmol) and exo − Klenow polymerase (5 U).
- ( B ) Amplification of a 102-bp target sequence with (lane 1) or without (lane 2) heat denaturation at 95°C, before a 2 h incubation at 37°C.
- A two-step HDA reaction, with a 10 min incubation at 95°C and 2 h incubation at 37°C, was performed.
- Another region (102 bp) of the earIR homologue gene was chosen as target.
- The yield of the one-step HDA amplification was about 40–60% of the two-step HDA reaction.
- First reported to comprise 10–12% ( McReynolds et al , 1986 ), and then 1% of the Brugia genome ( Ghedin et al , 2004 ), this highly repeated sequence became a target of choice for the detection of B.
- A specific product was detected for samples containing as low as 5 pg of B.
- malayi DNA, which corresponds to 500 copies of the genome ( Fig 4 ).
- malayi genomic DNA was added to 200 μl of human blood samples.
- After processing, 1 μl of each sample was used as template for HDA reactions.
- A two-step HDA reaction, with a 2 h incubation at 37°C, was performed.
- After 35 min, product accumulation generated a typical sigmoid curve.
- A semilogarithmic plot of the increase in fluorescence in the early phase of the reaction revealed an initial first-order reaction with a rate of amplification ( V ) of 0.23 RFU/min, which corresponds to a doubling time of 3 min ( Fig 5B ).
- Following the log-linear phase, the reaction slowed, entering a transition phase (between 45 and 80 min), eventually reaching the plateau phase ( Fig 5A ).
- Curves 1 and 2: two identical reactions performed on 10 ng of genomic DNA.
- However, its speed (20 bp/s) and processivity (less than 100 bp per binding) are limited ( Ali et al , 1999 ).
- Kong, unpublished data), which unwinds DNA at a rate of 300 bp/s and with high processivity ( Kornberg & Baker, 1992 ).
- The 10 × HDA buffer A contains 350 mM Tris-acetate (pH 7.5) and 100 mM dithiothreitol and the 10 × HDA buffer B contains 10 mM Tris-acetate (pH 7.5), 1 mg/ml bovine serum albumin and 100 mM magnesium acetate.
- HDA reaction component A (30 μl) was prepared by combining 5 μl of 10 × HDA buffer A, template (plasmid DNA, genomic DNA, cells, processed human blood sample (see supplementary information online for information on the preparation of the reconstituted human blood sample)), 10–20 pmol of each target-specific primer (see supplementary information online for details on the HDA primers), 20 nmol dNTPs and dH 2 O.

**工艺/辅料句（直引）**：
- In a first step, substrate DNA was mixed with the primers for heat denaturation and subsequent annealing.
- In the HDA reaction, the unwinding, primer-annealing and extension steps must be coordinated.

## pnas.0730811100
- 标题：Isothermal reactions for the amplification of oligonucleotides.
- 期刊/年：Proceedings of the National Academy of Sciences of the Unite 2003｜DOI: 10.1073/pnas.0730811100｜PMCID: PMC404692｜通道: -
- 本地全文：/Users/liuxinyuan/Desktop/Hermes输出-工作类/知识沉淀/_全文/OA/10.1073_pnas.0730811100.txt

**摘要（直引节选）**：Isothermal reactions for the amplification of oligonucleotides. PMCID: PMC404692 DOI: 10.1073/pnas.0730811100 Official websites use .gov A .gov website belongs to an official government organization in the United States. Secure .gov websites use HTTPS A lock ( Lock Locked padlock icon ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites. Search PMC Full-Text Archive Search in PMC Journal List User Guide PERMALINK Copy As a library, NLM provides access to scientific literature. Inclusion in an NLM database does not imply endorsement of, or agreement with, the contents by NLM or the National Institutes of Health. Learn more: PMC Disclaimer | PMC Copyright Notice Proc Natl Acad Sci U S A . 2003 Apr 4;100(8):4504–4509. doi: 10.1073/pnas.0730811100 Isothermal reactions for the amplification of oligonucleotides Jeffrey Van Ness Jeffrey Van Ness 1 Keck Graduate Institute of Applied Life Science, 535 Watson Drive, Claremont, CA 91711 Find articles by Jeffrey Van Ness 1 , Lori K Van Ness Lori K Van Ness 1 Keck Graduate Institute of Applied Life Science, 535 Watson Drive, Claremont, CA 91711 Find articles by Lori K Van

**含数值句（直引）**：
- Copyright © 2003, The National Academy of Sciences PMC Copyright notice PMCID: PMC404692 PMID: 12679520 Abstract We have devised a class of isothermal reactions for amplifying DNA.
- It is a highly sensitive chain reaction that can be specifically triggered by given DNA sequences and can achieve amplifications of >10 6 -fold.
- Another limitation of the rate of PCR derives from the nature of the reaction itself in that a maximum 2-fold amplification can be achieved in each cycle.
- Because of the balance between the thermal properties of the DNA oligonucleotides and the enzymes used, the optimum temperature of the reaction with these enzymes is 60°C (see Materials and Methods ).
- The primer template was present at 1 μM in a 50-μl reaction (see Materials and Methods ), and the yield of the reaction products is shown.
- The kinetic parameters used for the solution (see Materials and Methods ) were: r = 0.4 sec −1 ;
- The trigger oligonucleotide, σ, was present at 10 −5 μM at time 0.
- The fluorescence of SYBR green was monitored every 30 sec in six independent identical reactions.
- Bst NBI) has a specific activity of ≈10 6 units/mg (H.-M.
- The conditions for the linear reaction were: 85 mM KCl/25 mM Tris⋅HCl (pH 8.8, 25°C)/2.0 mM MgSO 4 /5 mM MgCl2/10 mM (NH4)2SO4/0.1% (vol/vol) Triton X-100/0.5 mM DTT/0.4 units/μl N.
- Bst NBI nicking enzyme/0.05 units/μl Vent exo − polymerase/400 μM dNTPs (Epicentre, Madison, WI)/10 μg/ml BSA/0.05 μM template and primer olignucleotides [NBtop and NB12 (equimolar) in ultrapure water that is nuclease-free (Ambion, Austin, TX)].
- Reactions were assembled at 4°C, initiated by transferring to a preheated thermocycler at 60°C, and stopped by incubation at 4°C.
- No further manipulations were performed before placement on the autoinjector for the HPLC-MS, which is held at 4°C.
- The exponential reactions were also carried out at 60°C, temperature controlled to within 0.1°C.
- The exponential reaction conditions were as follows: the same as described above for the linear reaction except with 0.1 μM template oligonucleotide only (unless otherwise noted).
- In the case of fluorescence monitoring, SYBR green (Molecular Probes) was added to 5× concentration (SYBR green is supplied by the manufacturer at 10,000×).
- The column is a Waters Xterra MS C18, incorporating C18 packing with 3.5 μM particle size, with 125-Å pore size, 2.1 mm × 20 mm.
- The column was run at 30°C with a gradient of acetonitrile in 5 mM dimethyl-butylamine acetate (DMBAA).
- As a check on the complete release of the signal oligo during the chromatography and injection, we ran the column at 50°C after incubating the sample briefly at 95°C.
- Buffer A is 5 mM DMBAA, and buffer B is 5 mM DMBAA and 50% (V/V) acetonitrile.
- A survey of a range of >80 different 12 mers indicated that the variation between sequences attributable to this difference is <30%.
- The real-time measurements on this instrument were made by using an isothermal protocol with a 30-sec interval read beginning 10 sec after the lid and chamber reached 60°C.
- The synthesized oligonucleotide is fully stable at 60°C when it is covalently joined to the rest of the upper stand, as it is immediately after its synthesis, but is only transiently stable as a 12 mer after the nicking reaction.
- The products of the reaction were analyzed on the LC-MS system after the indicated incubation times at 60°C (see Materials and Methods ).
- 1 , this initial rate is about one molecule (12 mer) per primer template every 2.5 sec, or ≈0.4 molecules per primer template ⋅ sec.
- Note that the reaction slows down noticeably after 10 min or so.

**工艺/辅料句（直引）**：
- The simplified mass action equations use the following variables: a , the annealing rate between the product oligonucleotide concentration, σ, and the amplification template concentration, τ;
- The equations, using the simplifying assumptions that annealing is a single-step bimolecular reaction and that the conversion of χ into π can be represented as a simple effective rate, are then, σ can easily be shown from these equations to exhibit exponential behavior.
- It seems likely that the diffusion and annealing of the product oligonucleotides to the amplification template is a slow step, judging from the diffusion constants of oligonucleotides in this molecular weight range.
- 4 depends for its specificity (that is, giving only the appropriate product) on a phenomenon that appears to be specific to the transient annealing and priming process that creates the primer templates.

## pnas.89.1.392
- 标题：Isothermal in vitro amplification of DNA by a restriction enzyme/DNA polymerase system.
- 期刊/年：Proceedings of the National Academy of Sciences of the Unite 1992｜DOI: 10.1073/pnas.89.1.392｜PMCID: PMC48243｜通道: -
- 本地全文：/Users/liuxinyuan/Desktop/Hermes输出-工作类/知识沉淀/_全文/OA/10.1073_pnas.89.1.392.txt

**摘要（直引节选）**：Isothermal in vitro amplification of DNA by a restriction enzyme/DNA polymerase system. PMCID: PMC48243 DOI: 10.1073/pnas.89.1.392 Official websites use .gov A .gov website belongs to an official government organization in the United States. Secure .gov websites use HTTPS A lock ( Lock Locked padlock icon ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites. Search PMC Full-Text Archive Search in PMC Journal List User Guide PERMALINK Copy As a library, NLM provides access to scientific literature. Inclusion in an NLM database does not imply endorsement of, or agreement with, the contents by NLM or the National Institutes of Health. Learn more: PMC Disclaimer | PMC Copyright Notice Proc Natl Acad Sci U S A . 1992 Jan 1;89(1):392–396. doi: 10.1073/pnas.89.1.392 Isothermal in vitro amplification of DNA by a restriction enzyme/DNA polymerase system. G T Walker G T Walker 1 Department of Molecular Biology, Becton Dickinson Research Center, Research Triangle Park, NC 27709. Find articles by G T Walker 1 , M C Little M C Little 1 Department of Molecular Biology, Becton Dickinson Research Center, Research Triangle Pa

**含数值句（直引）**：
- PMC Copyright notice PMCID: PMC48243 PMID: 1309614 Abstract An isothermal in vitro DNA amplification method was developed based upon the following sequence of reaction events.
- A 10(6)-fold amplification of a genomic sequence from Mycobacterium tuberculosis or Mycobacterium bovis was achieved in 4 h at 37 degrees C.

## pnas.92.10.4641
- 标题：Rolling replication of short DNA circles.
- 期刊/年：Proceedings of the National Academy of Sciences of the Unite 1995｜DOI: 10.1073/pnas.92.10.4641｜PMCID: PMC42000｜通道: -
- 本地全文：/Users/liuxinyuan/Desktop/Hermes输出-工作类/知识沉淀/_全文/OA/10.1073_pnas.92.10.4641.txt

**摘要（直引节选）**：Rolling replication of short DNA circles. PMCID: PMC42000 DOI: 10.1073/pnas.92.10.4641 Official websites use .gov A .gov website belongs to an official government organization in the United States. Secure .gov websites use HTTPS A lock ( Lock Locked padlock icon ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites. Search PMC Full-Text Archive Search in PMC Journal List User Guide PERMALINK Copy As a library, NLM provides access to scientific literature. Inclusion in an NLM database does not imply endorsement of, or agreement with, the contents by NLM or the National Institutes of Health. Learn more: PMC Disclaimer | PMC Copyright Notice Proc Natl Acad Sci U S A . 1995 May 9;92(10):4641–4645. doi: 10.1073/pnas.92.10.4641 Rolling replication of short DNA circles. A Fire A Fire 1 Department of Embryology, Carnegie Institution of Washington, Baltimore, MD 21210, USA. Find articles by A Fire 1 , S Q Xu S Q Xu 1 Department of Embryology, Carnegie Institution of Washington, Baltimore, MD 21210, USA. Find articles by S Q Xu 1 Author information Copyright and License information 1 Department of Embryology, Carnegie I

**含数值句（直引）**：
- PMC Copyright notice PMCID: PMC42000 PMID: 7753856 Abstract Natural genes and proteins often contain tandemly repeated sequence motifs that dramatically increase physiological specificity and activity.
- In this communication, we demonstrate that rolling circle synthesis in a simple enzymatic system can produce tandem repeats of monomers as short as 34 bp.

## 2021.04.15.439918
- 标题：Multi-modal engineering of Bst DNA polymerase for thermostability in ultra-fast LAMP reactions
- 期刊/年：bioRxiv (Cold Spring Harbor Laboratory) 2021｜DOI: 10.1101/2021.04.15.439918｜PMCID: ｜通道: -
- 本地全文：/Users/liuxinyuan/Desktop/Hermes输出-工作类/知识沉淀/_全文/OA/10.1101_2021.04.15.439918.txt

**摘要（直引节选）**：DNA polymerase from Geobacillus stearothermophilus, Bst DNA polymerase (Bst DNAP), is a versatile enzyme with robust strand-displacing activity that enables loop-mediated isothermal amplification (LAMP). Despite its exclusive usage in LAMP assay, its properties remain open to improvement. Here, we describe logical redesign of Bst DNAP by using multimodal application of several independent and orthogonal rational engineering methods such as domain addition, supercharging, and machine learning predictions of amino acid substitutions. The resulting Br512g3 enzyme is not only thermostable and extremely robust but it also displays improved reverse transcription activity and the ability to carry out ultrafast LAMP at 74 °C. Our study illustrates a new enzyme engineering strategy as well as contributes a novel engineered strand displacing DNA polymerase of high value to diagnostics and other fields.

**含数值句（直引）**：
- 1 Multi-modal engineering of Bst DNA polymerase for thermostability in ultra-fast LAMP reactions Inyup Paik1,2, Phuoc H.
- The resulting Br512g3 enzyme is not only thermostable and extremely robust but it also displays improved reverse transcription activity and the ability to carry out ultrafast LAMP at 74 °C.
- In addition, HP35 displays thermostability with a transition midpoint (Tm) of 70°C (6,7).
- A single colony was seed cultured overnight in 5 mL of superior broth (Athena Enzyme Systems, 0105).
- The next day, 1 mL of seed culture was inoculated into 1 L of superior broth and grown at 37 OC until it reached an OD600 of 0.5-0.6.
- Enzyme expression was induced with 1 mM IPTG and 100 ng/mL of anhydrous tetracycline (aTc) at 18 OC for 18 h (or overnight).
- The induced cells were pelleted at 5000xg for 10 min at 4OC and resuspended in 30mL of ice-cold lysis buffer (50 mM Phosphate Buffer, pH 7.5, 300 mM NaCl, 20 mM imidazole, 0.1% Igepal CO-630, 5 mM MgSO4, 1 mg/mL HEW Lysozyme, 1x EDTA-free protease inhibitor tablet, Thermo Scientific, A32965).
- The samples were then sonicated (1 sec ON, 4 sec OFF) for a total time of 4 minutes with 40% amplitude.
- The lysate was centrifuged at 35,000 xg for 30 minutes at 4 °C.
- The supernatant was transferred to a clean tube and filtered through a 0.2 μm filter.
- Briefly, 1mL of Ni-NTA agarose slurry was packed into a 10mL disposable column and equilibrated with 20 column volume (CV) of equilibration buffer (50 mM Phosphate Buffer, pH 7.5, was not certified by peer review) is the author/funder.
- https://doi.org/10.1101/2021.04.15.439918 doi: bioRxiv preprint 3 300 mM NaCl, 20 mM imidazole).
- Following loading, the column was washed with 20 CV of equilibration buffer and 5 CV of wash buffer (50 mM Phosphate Buffer, pH 7.5, 300 mM NaCl, 50 mM imidazole).
- Br512 was eluted with 5 mL of elution buffer (50 mM Phosphate Buffer, pH 7.5, 300 mM NaCl, 250 mM imidazole).
- The eluate was dialyzed twice with 2L of Ni-NTA dialysis buffer (40 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM DTT, 0.1% Igepal CO-630).
- The dialyzed eluate was further passed through an equilibrated 5 mL heparin column (HiTrap™ Heparin HP) on a FPLC (AKTA pure, GE healthcare) and eluted using a linear NaCl gradient generated from heparin buffers A and B (40 mM Tris-HCl, pH 7.5, 100 mM NaCl for buffer A;
- 2M NaCl for buffer B, 0.1% Igepal CO-630).
- The collected final eluate was dialyzed first with 2 L of heparin dialysis buffer (50 mM Tris-HCl, pH 8.0, 50 mM KCl, 0.1% Tween-20) and second with 2 L of final dialysis buffer (50% Glycerol, 50 mM Tris-HCl, pH 8.0, 50 mM KCl, 0.1% Tween-20, 0.1% Igepal CO-630, 1 mM DTT).
- Real-time GAPDH LAMP-OSD LAMP-OSD reaction mixtures were prepared in 25 µL volume containing indicated amounts of human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) DNA templates along with a final concentration of 1.6 μM each of BIP and FIP primers, 0.4 μM each of B3 and F3 primers, and 0.8 μM of the loop primer.
- The buffer was appended with 1 M betaine, 0.4 mM dNTPs, 2 mM additional MgSO4 (only for reactions in Isothermal buffer), and either Bst 2.0 DNA polymerase (16 units), Bst-LF DNA polymerase (20 pm), or Br512 DNA polymerase (0.2 pm, 2 pm, 20 pm, or 200 pm).
- Assays read using OSD probes received 100 nM of OSD reporter prepared by annealing 100 nM fluorophore-labeled OSD strands with a 5-fold excess of the quencher-labeled OSD strands by incubation at 95 °C for 1 min followed by cooling at the rate of 0.1 °C/sec to 25 °C.
- For real-time signal measurement these LAMP reactions were transferred into a 96-well PCR plate, which was incubated in a LightCycler 96 real-time PCR machine (Roche, Basel, Switzerland) maintained at 65 °C for 90 min.
- Fluorescence signals were recorded every 3 min in the FAM channel and analyzed using the LightCycler 96 software.
- Endpoint GAPDH LAMP-OSD LAMP-OSD reaction mixtures for visual endpoint readout of OSD fluorescence were prepared in 25 µL volume containing either 1X Isothermal buffer and 16 units of Bst 2.0 or G6B buffer and 20 pm of Br512.
- The reactions also contained 1 M betaine, 1.4 mM dNTPs, 2 mM additional MgSO4 (only for reactions in Isothermal buffer), and 100 nM OSD reporter prepared by annealing 100 nM fluorophore-labeled OSD strands with a 2-fold excess of the quencher-labeled OSD strands by incubation at 95 °C for 1 min followed by cooling at the rate of 0.1 °C/sec to 25 °C.
- A final concentration of 1.6 μM each of BIP and FIP primers, 0.4 μM each of B3 and F3 primers, and 0.8 μM of the loop primer were added to some reactions while control assays without primers received the same volume of water.

**工艺/辅料句（直引）**：
- The purified Br512 was quantified by Bradford assay and SDS-PAGE/coomassie gel staining alongside a bovine serum albumin (BSA) standard.
- First, Tholoth, Lamb, and NB OSD probes were prepared by annealing 1 µM of the fluorophore-labeled OSD strand with 2 µM, 3 µM, and 5 µM, respectively of the quencher-labeled strand in 1X Isothermal buffer.
- Annealing was performed by denaturing the oligonucleotide mix at 95 °C for 1 min followed by slow cooling at the rate of 0.1 °C/s to 25 °C.
- https://doi.org/10.1101/2021.04.15.439918 doi: bioRxiv preprint 5 fold excess of the quencher labeled OSD strand, (vi) 1.25 micromoles of trehalose, and (vii) 20 pm or 30 pm of BR512.
- The reagent mixes were distributed in 0.2 mL PCR tubes and frozen for 1 h on dry ice prior to lyophilization for 2.5 h at 197 mTorr and -108 °C using the automated settings in a VirTis Benchtop Pro lyophilizer (SP Scientific, Warminster, PA, USA).
- Lyophilized assays were stored with desiccant at -20 °C until use.
- Lyophilized assays were rehydrated immediately prior to use by adding 22 µL of 1X G6D buffer containing 10 micromoles of betaine.

## gr.180501
- 标题：Rapid amplification of plasmid and phage DNA using Phi 29 DNA polymerase and multiply-primed rolling circle amplification.
- 期刊/年：Genome research 2001｜DOI: 10.1101/gr.180501｜PMCID: PMC311129｜通道: -
- 本地全文：/Users/liuxinyuan/Desktop/Hermes输出-工作类/知识沉淀/_全文/OA/10.1101_gr.180501.txt

**摘要（直引节选）**：Rapid amplification of plasmid and phage DNA using Phi 29 DNA polymerase and multiply-primed rolling circle amplification. PMCID: PMC311129 DOI: 10.1101/gr.180501 Official websites use .gov A .gov website belongs to an official government organization in the United States. Secure .gov websites use HTTPS A lock ( Lock Locked padlock icon ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites. Search PMC Full-Text Archive Search in PMC Journal List User Guide PERMALINK Copy As a library, NLM provides access to scientific literature. Inclusion in an NLM database does not imply endorsement of, or agreement with, the contents by NLM or the National Institutes of Health. Learn more: PMC Disclaimer | PMC Copyright Notice Genome Res . 2001 Jun;11(6):1095–1099. doi: 10.1101/gr.180501 Rapid Amplification of Plasmid and Phage DNA Using Phi29 DNA Polymerase and Multiply-Primed Rolling Circle Amplification Frank B Dean Frank B Dean 1 Molecular Staging, Inc., New Haven, Connecticut 06511, USA; 2 Amersham Pharmacia Biotech, Piscataway, New Jersey 08855-1327, USA Find articles by Frank B Dean 1, 3 , John R Nelson John R Nelson

**含数值句（直引）**：
- Copyright © 2001, Cold Spring Harbor Laboratory Press PMC Copyright notice PMCID: PMC311129 PMID: 11381035 Abstract We describe a simple method of using rolling circle amplification to amplify vector DNA such as M13 or plasmid DNA from single colonies or plaques.
- Using random primers and φ29 DNA polymerase, circular DNA templates can be amplified 10,000-fold in a few hours.
- Previously, RCA had been used to amplify small DNA circles approximately 100 nt in length.
- However, the rate for plasmid-sized targets is only about 20 copies per hour, limiting the usefulness with plasmids or other circles larger than 0.2 kb.
- We describe here a technique called multiply-primed rolling circle amplification (multiply-primed RCA) that uses the unique properties of φ29 DNA polymerase and random primers to achieve a 10,000-fold amplification.
- The method is simple and is optimally performed at 30°C, making it suitable for a variety of applications.
- φ29 DNA polymerase was chosen because of its capacity to perform strand displacement DNA synthesis for more than 70,000 nt without dissociating from the template ( Blanco et al.
- The M13 DNA was amplified 375-fold in 24 h by using random hexamers.
- Rolling circle reactions were performed as described and contained either 1 ng of singly-primed single-strand M13 DNA or 1 ng of random hexamer-primed single-strand M13 DNA.
- Reactions were incubated at 34°C for 24 h and aliquots were taken to measure DNA synthesis as indicated.
- Approximately 80%of the multiply-primed RCA products on the gel were converted to the linear form by Eco RI digestion.
- For 200 copies of pUC19, genomic DNA would be in an eightfold excess in the cell.
- Up to a 10,000-fold amplification was achieved starting with 1 ng of M13 template.
- This compared favorably with the 375-fold amplification observed by using exo-sensitive primers (Fig.
- It corresponded to an amplification rate of ∼800 copies per hour, a 40-fold improvement over the rate of 20 copies per hour achieved with linear RCA.
- 4 , inset) confirmed the improvement in yield using exo-resistant primers and showed the average product length to be greater than 40 kb.
- These results are consistent with the observation that, in assays containing more than 0.3 units of φ29 DNA polymerase per nanogram of DNA using unmodified primers, the amplification yield was decreased (Fig.
- Annealing reactions (20 μL) were performed as described except that the template DNA was M13 double-strand RF DNA and the primers were either exo-resistant or exo-sensitive hexamers.
- Reactions contained 1 ng of M13 DNA with exo-resistant or exo-sensitive primers and φ29 DNA polymerase as indicated.
- Reactions were incubated at 34°C for 13 h.
- The quality and read length of the sequence was indistinguishable from that obtained with ∼100 ng DNA template purified by standard methods.
- A saturated culture of XL1-blue (Stratagene) containing a random library plasmid (2- to 3-kb inserts in pUC18, 2 μL) was amplified as described for 12 h (see Methods).
- The amplified DNA was treated with calf intestine phosphatase, heated to 95° for 3 min, and sequenced by using the DYEnamic ET terminator sequencing kit and run on a MegaBACE 1000 DNA sequencer.
- An important factor for the success of this method is the unique nature of φ29 DNA polymerase: This single subunit, proofreading DNA polymerase, is able to incorporate >70,000 nt per binding event ( Blanco et al.
- It has excellent strand displacement activity and is very stable, with linear reaction kinetics at 30°C for over 12 h.
- The use of random hexamer primers with 3′ thiophosphate-protected ends is also important, allowing circular DNA molecules to be amplified at least 10,000-fold by protecting the primers from the 3′ exonuclease activity of φ29 DNA polymerase.

**工艺/辅料句（直引）**：
- The heating step inactivates nucleases, releases the plasmid or phage DNA from cells or phage particles, and denatures the DNA, allowing primer annealing.
- For preparation of random hexamer-primed M13 DNA, annealing reactions (60 μL) contained 20 mM Tris-HCl (pH 7.5), 20 mM KCl, 0.1 mM ethylenediaminetetraacetic acid (EDTA), 15 fmoles (35 ng) of single-strand M13 circles, and 6000 pmoles random hexamer.

## journal.pbio.0040204
- 标题：DNA Detection Using Recombination Proteins
- 期刊/年：PLoS Biology 2006｜DOI: 10.1371/journal.pbio.0040204｜PMCID: ｜通道: -
- 本地全文：/Users/liuxinyuan/Desktop/Hermes输出-工作类/知识沉淀/_全文/OA/10.1371_journal.pbio.0040204.txt

**摘要（直引节选）**：DNA Detection Using Recombination Proteins Olaf Piepenburg1, Colin H. Williams1, Derek L. Stemple2, Niall A. Armes1* 1 ASM Scientific Ltd, Cambridge, United Kingdom, 2 Wellcome Trust Sanger Institute, Hinxton, Cambridge, United Kingdom DNA amplification is essential to most nucleic acid testing strategies, but established techniques require sophisticated equipment or complex experimental procedures, and their uptake outside specialised laboratories has been limited. Our novel approach, recombinase polymerase amplification (RPA), couples isothermal recombinase-driven primer targeting of template material with strand-displacement DNA synthesis. It achieves exponential amplification with no need for pretreatment of sample DNA. Reactions are sensitive, specific, and rapid and operate at constant low temperature. We have also developed a probe-based detection system. Key aspects of the combined RPA amplification/detection process are illustrated by a test for the pathogen methicillin-resistant Staphylococcus aureus. The technology proves to be sensitive to fewer than ten copies of genomic DNA. Furthermore, products can be detected in a simple sandwich assay, thereby establishing an inst

**含数值句（直引）**：
- Published June 13, 2006 DOI: 10.1371/journal.pbio.0040204 Copyright: 
 2006 Piepenburg et al.
- The amplification of DNA proved to be exponen- tial over a wide range of template concentrations and results were obtained in less than 30 min.
- RPA Amplifies Specific Target Regions from Complex DNA Templates in less than 30 Minutes (A) Acrylamide gel electrophoresis of RPA products using primers for three human markers (apoB, Sry, PBDG).
- Water (
) or 1,000 copies of genomic DNA (þ) served as template.
- subtilis DNA served as template in triplicate reactions at 105 (black), 104 (red), 103 (yellow), or 100 copies (green) or water (blue).
- The onset of amplification at identical starting template quantities of ten, 100, 1,000, or 10,000 copies was virtually simultaneous, demonstrating the robustness of the technique.
- 104 copies) serving as a negative control did not generate a signal, despite being amplified by suitable primers (see Figure S5).
- Reactions were performed at 37 8C for 60 min or as indicated.
- Standard conditions were 50 mM Tris (pH 7.9), 100 mM potassium acetate, 14 mM magnesium acetate, 2 mM DTT, 5% Carbowax20M, 200 lM dNTPs, 3 mM ATP, 50 mM phosphocreatine, 100 ng/ll creatine kinase, and 30 ng/ll Bsu;
- in the multiplex experiment, Carbowax20M was at 5.5%.
- Recombinase protein concentrations were 900 ng/ll gp32, 120 ng/ll uxsX, and 30 ng/ll uvsY.
- Primers used for amplification of human targets were apoB4, apoB300, sry3, sry4, pbdg5, pbdg6 (300 nM each), for B.
- subtilis, bs1 and bs2 were used (300 nM each) in the experiment in Figure 2B.
- For MRSA amplification, primers used were 500 nM sccIII, 100 nM orfX (Figure 4A) or 265 nM sccI/II, 265 nM sccIII, 70 nM orfX (Figure 5) or 240 nM Bio-sccI/II, 240 nM Bio-sccIII, 120 nM orfX (Figure 6).
- A Multiplex RPA Approach Enables Detection of Different MRSA Alleles and an Internal Control in the Same Reaction (A) MRSAI (green), MRSAII (dark blue), MRSAIII DNA (red) at ten copies, or MSSA DNA at 104 copies (blue, negative control) or water (yellow, turquoise, negative controls) served as a template (in triplicate for each template condition).
- (B) Detection of 50 copies of internal control DNA included in the reactions in (A).
- Probes were used at 60 nM SATamra1 (MRSAIII experiment) or at 45 nM SATamra1, 45 nM SATamra2, 60 nM BSFlc (multiplex experiment).
- Excitation/detection was at 485/525 nm (SybrGreenI, BSFlc) or 530/575 nm (SATamra1/2).
- Measurements were taken every 30 or 45 s (multiplex experiment).
- Reactions contained (left to right) ten copies of MRSAIII, ten copies of MRSAII, ten copies of MRSAI, or 10,000 copies of MSSA (negative control) as template.
- For lateral-flow strip experiments, two probes were used at 75 nM each: Lfs1 59-FAM-ccacatcaaatgatgcgggttgtgttaat(H)gaacaagtgtacagag- ddC-39 Lfs2 59-FAM-ccacatcaaatgatgcgggttgtgttaat(H)gagcaagtgtatagag- ddC-39 The 59-biotinylated forms of sccI/II and sccIII were utilised as primers.
- For each reaction (20 ll), 1 ll was diluted with 5 ll of running buffer (PBS/3% Tween) and applied directly to HybriDetect strips (Milenia Biotec, Bad Nauheim, Germany) according to the manufacturer’s instructions.
- Reactions were performed at 37 8C for 120 min and contained the primers sry3 and sry4 at 300 nM, 50 mM Tris (pH 8.4), 80 mM potassium acetate, 10 mM magnesium acetate, 2 mM DTT, 3 mM ATP, 200 lM dNTPs, 20 mM phosphocreatine, 100 ng/ll creatine kinase, 5% Carbowax20M, 600 ng/ll gp32, 200 ng/ll uvsX, 60 ng/ll uvsY, and 30 ng/ll Bsu, except when a given component was that under investigation.
- And 1,500 copies/ll of the human Y-chromosomal DNA served as template in 30-ll reactions (per sample the equivalent of 10-ll reaction volume was loaded on the gel).
- Found at DOI: 10.1371/journal.pbio.0040204.sg001 (159 KB PDF).
- Reactions were performed at 37 8C for 120 min, and conditions used were 50 mM Tris (pH 8.4), 80 mM potassium acetate, 10 mM magnesium acetate, 2 mM DTT, 3 mM ATP, 200 lM dNTPs, 20 mM phosphocreatine, 100 ng/ll creatine kinase, 5% Carbowax20M, 600 ng/ ll gp32, 125 ng/ll uvsX, 25 ng/ll uvsY, and 30 ng/ll Bsu.

**工艺/辅料句（直引）**：
- Coding sequences for uvsx, uvsy, gp32, Bsu, and Nfo were amplified from genomic DNA (DSMZ, Braunschweig Germany), fused to hexahistidine-tags (N-terminal for uvsY, Bsu, and Nfo, C-terminal for uvsX and gp32), and cloned into suitable expression vectors.

## clinchem.2015.245829
- 标题：Recombinase Polymerase Amplification for Diagnostic Applications.
- 期刊/年：Clinical chemistry 2016｜DOI: 10.1373/clinchem.2015.245829｜PMCID: PMC7108464｜通道: -
- 本地全文：/Users/liuxinyuan/Desktop/Hermes输出-工作类/知识沉淀/_全文/OA/10.1373_clinchem.2015.245829.txt

**摘要（直引节选）**：Recombinase Polymerase Amplification for Diagnostic Applications. PMCID: PMC7108464 DOI: 10.1373/clinchem.2015.245829 Official websites use .gov A .gov website belongs to an official government organization in the United States. Secure .gov websites use HTTPS A lock ( Lock Locked padlock icon ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites. Search PMC Full-Text Archive Search in PMC Journal List User Guide PERMALINK Copy As a library, NLM provides access to scientific literature. Inclusion in an NLM database does not imply endorsement of, or agreement with, the contents by NLM or the National Institutes of Health. Learn more: PMC Disclaimer | PMC Copyright Notice Clin Chem . 2016 Jul 1;62(7):947–958. doi: 10.1373/clinchem.2015.245829 Recombinase Polymerase Amplification for Diagnostic Applications Rana K Daher Rana K Daher 1 Centre de recherche en infectiologie de l'Université Laval (CRI), Axe maladies infectieuses et immunitaires, Centre de recherche du CHU de Québec-Université Laval, Québec City (Québec), Canada 2 Département de microbiologie-infectiologie et d'immunologie, Faculté de médecine, Univers

**含数值句（直引）**：
- PMC Copyright notice PMCID: PMC7108464 PMID: 27160000 Abstract BACKGROUND First introduced in 2006, recombinase polymerase amplification (RPA) has stirred great interest, as evidenced by 75 publications as of October 2015, with 56 of them just in the last 2 years.
- The widespread adoption of this isothermal molecular tool in many diagnostic fields represents an affordable (approximately 4.3 USD per test), simple (few and easy hands-on steps), fast (results within 5–20 min), and sensitive (single target copy number detected) method for the identification of pathogens and the detection of single nucleotide polymorphisms in human cancers and genetically modified organisms.
- Owing to minimal sample-preparation requirements, low operation temperature (25–42 °C), and commercial availability of freeze-dried reagents, this method has been applied outside laboratory settings, in remote areas, and interestingly, onboard automated sample-to-answer microfluidic devices.
- In this review, we focus on recombinase polymerase amplification (RPA) 3 because of its simplicity (few and easy hands-on manipulations), flexibility (different commercial kit formats offering various detection methods for both DNA and RNA), and speed (results in 5–20 min).
- RPA has high specificity and efficiency (10 4 -fold amplification in 10 min) ( 4 ) and displays a completely isothermal profile not requiring an additional temperature step for DNA denaturation.
- The complete reaction is performed at a single temperature from 25–42 °C depending on RPA kit formulation.
- Typically, RPA reactions are executed at a single temperature (37 °C) in 5–20 min depending on the starting template copy number and amplicon size ( 6 ).
- 1-step reverse-transcriptase RPA (RT-RPA) operates at a single temperature (40–42 °C for exo RT or basic RT kits, respectively) while still enabling target detection in 20 min ( 7 ).
- A typical RPA kit comprises individual tubes containing lyophilized RPA reagent pellets, 1× reaction buffer, magnesium acetate, and controls (positive control DNA with control primers and probe).
- RPA kits can be stored for up to 6 months at −20 °C before use.
- Incubation is performed at a single temperature ranging between 37 and 42 °C depending on the kit used ( Table 1 ).
- With real-time detection, results are obtained during the incubation stage, typically in <10 min.
- With lateral-flow detection, RPA amplicons can be used directly without purification, generating results on the dipstick strip within 5 min post-RPA.
- Low or high GC content (<30% or >70%) must also be avoided.
- RPA can amplify long sequences up to 1.5 kb;
- however, better results are obtained with shorter amplicons in the range of 80–400 bp (100–200 bp optimal) ( 4 ).
- Fluorescence generation typically yields a detectable signal within 5–10 min during the RPA reaction ( 6 ).
- By analyzing a total of 204 RPA primers and 64 probe [exo (75%), fpg (14%), and nfo (11%) probes] sequences originating from 40 of the 75 published RPA articles, we observed that some authors have succeeded in using RPA primers/probes presenting some differences from the manufacturer guidelines.
- For instance, 9% of RPA primers were below 30 bases and 7% were over 35 bases long (36–45 bases).
- Ten percent of primers sequences had a G+C content <30% and 3% had a G+C content >65%.
- For published exo probes sequences, 17% of exo probes were shorter than the recommended length (34–44 bases instead of 46–52 bases) and 10% were of 53 and 58 bases long.
- A small proportion (6%) of exo probes had G+C content below 30%.
- Most of the probes that were considered in our review used the exo probe real-time detection method (75%) rather than fpg (14%) or nfo (11%) probes.
- The analytical limit of detection and the turnaround time varied between assays: for RNA detection, 10–21 copies of target detected in 2–20 min and 100–5 × 10 4 copies in 4–20 min;
- for DNA detection, 1–50 copies detected in 7–35 min and 98–778 copies detected in 8–35 min, suggesting that RPA efficiency could be dependent on target sequence, amplicon size, and type of biological sample tested.
- ( 25 ) Schmallenberg virus Blood/serum 6 × 10 3 <10 rt — Aebischer et al.

**工艺/辅料句（直引）**：
- For DNA unwinding and primer annealing, RPA uses recombinase enzymes with accessory proteins.
- It is possible to incorporate target-specific RPA primers/probe to the lyophilized RPA reagent pellet ( 9 ).
- This mixture is then added directly to 1 lyophilized RPA reagent pellet followed by brief mixing and centrifugation steps.
- In addition, there are no melting temperature requirements for RPA primers and probes because primer annealing and elongation are enzyme mediated and not temperature driven.
- For RNA detection, several groups ( 7 , 9 , 27 , 29 , 31 ) have used an exo RT kit that contained murine leukemia virus (MuLV) RT in the lyophilized pellet mixture ( 7 , 25 , 42 , 43 ).
- The cartridge allowed the conduction of 30 parallel RPA reactions and the disc contained 6 chambers having 5 cavities and each chamber was prestored with lyophilized RPA reagents.
- Currently, the cost of a RPA reaction is estimated to be $4.3 USD ( 51 ) and TwistDx offers a custom freeze-drying service for RPA reaction pellets, to facilitate the use and market penetration of RPA.

## fsens.2021.752600
- 标题：Isothermal Amplification of Nucleic Acids: The Race for the Next “Gold Standard”
- 期刊/年：Frontiers in Sensors 2021｜DOI: 10.3389/fsens.2021.752600｜PMCID: ｜通道: -
- 本地全文：/Users/liuxinyuan/Desktop/Hermes输出-工作类/知识沉淀/_全文/OA/10.3389_fsens.2021.752600.txt

**摘要（直引节选）**：Isothermal Amplification of Nucleic Acids: The Race for the Next “Gold Standard” Beatriz B. Oliveira 1,2, Bruno Veigas 3 and Pedro Viana Baptista 1,2* 1Nanomedicine Group, UCIBIO, Dept, Ciências da Vida, Faculdade de Ciências e Tecnologia, Universidade NOVA de Lisboa, Caparica, Portugal, 2i4HB, Associate Laboratory—Institute for Health and Bioeconomy, Faculdade de Ciências e Tecnologia, Universidade NOVA de Lisboa, Caparica, Portugal, 3ALMASCIENCE, Caparica, Portugal Nucleic acid amplification technologies (NAATs) have become fundamental tools in molecular diagnostics, due to their ability to detect small amounts of target molecules. Since its development, Polymerase Chain Reaction (PCR) has been the most exploited method, being stablished as the “gold standard” technique for DNA amplification. However, the requirement for different working temperatures leads to the need of a thermocycler machine or complex thermal apparatus, which have been preventing its application in novel integrated devices for single workflow and high throughput analysis. Conversely, isothermal amplification methods have been gaining attention, especially for point-of-care diagnosis and applications. These non-PCR

**含数值句（直引）**：
- Among the publications concerning these amplification techniques, most are focused on the analysis of water and food, with LAMP covering 67 and 59% of the publications, respectively.
- Then RPA covers for circa 20%, and the remainders, RPA, RCA, HDA and NASBA add up to 5%.
- and 2) cycling, where the dsDNA molecules are actively transcribed into RNA products, leading to a yield of 10–100 copies of RNA from each template molecule (Hønsvall and Robertson 2017) (Figure 3A).
- Even though this mechanism requires three enzymes - Reverse transcriptase, RNase H and T7 RNA polymerase, there are some advantages when compared to Reverse-transcription-PCR (RT- PCR), such as isothermal working temperature (41°C), which allows amplification on a simple heating block, shorter reaction FIGURE 2 | Number of scientific publications per year concerning amplification systems from 2000 until 2021.
- Because of thiol-modified dATP, only the original primer is cleaved by HincII but not the newly synthetized strand, resulting in 3’ overhangs, which are extended by the exonuclease deficient klenow (exo-klenow) polymerase and further displace the downstream DNA strand, at temperatures ranging from 30—50°C.
- Despite the advantages of isothermal reaction, the low temperature (between 30 and 55°C) makes it prone to unspecific primer-binding that might cause unspecific amplifications (Karami et al., 2011), similarly to what happens with NASBA.
- The high amplification efficiency of SDA associated to the capability to amplify small fragments (ranging from 50 to 120 bp) has highlighted its potential for miRNA expression profiling (Ye et al., 2019;
- Still, SDA takes a long-time to detection (2.5 h), which makes SDA inadequate for on-site amplifications and detection.
- Additionally, offers medium-free transport for swabs and room temperature storage and the total time of the assays ranges from 3 to 4 h (Van Der Pol et al., 2001).
- Compared to other NAATs, LAMP is 10 to 100-fold more sensitive than PCR (X.
- Wang X et al., 2014), with a detection limit as low as 1 copy per µL of template (H.
- Zhang et al., 2019), showing a higher specificity (Fujino et al., 2005) with an amplification time usually less than 1 h at 60–65°C, yielding 109 copies (Notomi et al., 2000).
- Additional advantages include not requiring an initial template denaturation step at 95°C and being less prone to inhibitory substances often present in biological samples (Enomoto et al., 2005;
- The cycling reaction continues leading to an accumulation of around 109 copies of target under the form of concatemers in less than 1 h (Notomi et al., 2000) (Figure 3C).
- Some of the available commercially and certified detections assays take between 15 and 30 min to result.
- Since the development of the first microfluidic device for the detection of hepatitis B virus, that accomplished a sensitivity of about 50 copies per reaction within 60 min (S.
- Recombinase Polymerase Amplification Recombinase Polymerase Amplification (RPA) is an isothermal amplification mechanism that operates at low temperature, usually between 37°C and 42°C, which exploits the activity of two different enzymes: a recombinase and a DNA polymerase and single stranded DNA-binding proteins.
- RPA allows amplification to occur in 20—40 min, exhibiting a detection limit of 1 copy of target, being considered as one of the fastest NAATs available (de Paz et al., 2014).
- These RPA-based assays attain comparable sensitivities to those obtained with PCR, but with a significant reduction in the reaction time—time to full reaction between 10 and 20 min (Kunze et al., 2016).
- Moreover, RPA assays show a detection limit around 10–20 copies of target, reduced sample and reagent volumes, and the capability to amplify under high concentrations of known PCR inhibitors (Polymerase and Rpa 2017;
- For example, Rohrman and Richards-Kortum accomplished the detection of 10 copies of HIV DNA in under 15 min through RPA incorporated with a lateral flow assay allowing to perform HIV diagnostics compatible with resource poor settings (B.
- Additionally, an RPA assay was also developed for the detection of Tuberculosis under 20 min with a limit of detection in the femtograms of template (Boyle et al., 2014).
- Rolling Circle Amplification Rolling Circle Amplification (RCA) is an isothermal amplification protocol that operates at low temperature (23°C—60°C), which has been adapted from the in vivo RCA mechanism (Fire and Xu, 1995).
- The RCA architecture offers two main benefits: 1) the low temperature isothermal scheme, with incubation temperatures as low as 23°C;
- and 2) the simplicity of its mechanism requirements (single primer and a productive DNA polymerase) (Li and Macdonald 2015), which yields high amplification efficiencies (∼103 copies in under 1 h), that could be enhanced though the addition of ssDNA- binding proteins (Y.
- (2012) DNA amplification M13 phage and pUC16 Multiply primed RCA Gel electrophoresis Dean et al.

**工艺/辅料句（直引）**：
- While PCR requires the lowering and rising of two or three working temperatures, to allow annealing and denaturation of DNA strands and primers, and to facilitate strand displacement, IA methods proceed at a single working temperature, which removes the need for a thermocycler.
- The DNA synthesis is initiated by the DNA polymerase upon the annealing of the outer primer (F3) to its complementary region and its subsequent extension causes strand displacement, releasing a ssDNA molecule.
- This molecule serves as template for the annealing on the other end of the second inner (BIP) and outer (B3) primers, producing a stem-loop DNA structure.
- coli or as usually used uvsX from T4-like bacteriophages) to primers in the presence of ATP and a crowding agent (high molecular weight PEG or Carbowax20M), leading to the formation of a recombinase-primer complex.