参见作者原研究论文

本实验方案简略版
Aug 2020

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Colorimetric RT-LAMP and LAMP-sequencing for Detecting SARS-CoV-2 RNA in Clinical Samples
比色RT-LAMP和LAMP测序检测临床样本中SARS-CoV-2 RNA   

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Abstract

During pandemics, such as the one caused by SARS-CoV-2 coronavirus, simple methods to rapidly test large numbers of people are needed. As a faster and less resource-demanding alternative to detect viral RNA by conventional qPCR, we used reverse transcription loop-mediated isothermal amplification (RT-LAMP). We previously established colorimetric RT-LAMP assays on both purified and unpurified SARS-CoV-2 clinical specimens and further developed a multiplexed sequencing protocol (LAMP-sequencing) to analyze the outcome of many RT-LAMP reactions at the same time (Dao Thi et al., 2020). Extending on this work, we hereby provide step-by-step protocols for both RT-LAMP assays and read-outs.

Keywords: RT-LAMP (逆转录环介导等温扩增), LAMP-sequencing (环介导等温扩增测序), SARS-CoV-2 detection (SARS-CoV-2检测), Tn5 tagmentation (Tn5测序建库方法), Colorimetric assay (比色测定)

Background

The new SARS-CoV-2 coronavirus poses a major public health problem (reviewed in Li et al., 2020). In the absence of efficient antiviral treatments and a protective vaccine, preventing local outbreaks by mass testing is critical. The standard diagnostic pipeline to detect SARS-CoV-2 infections is based on the isolation of viral RNA from clinical specimens, a reverse-transcription (RT) reaction to transcribe the RNA into cDNA, and detection by a semi-quantitative DNA polymerase chain reaction (qPCR) (Corman et al., 2020). Yet, commercial RNA isolation and RT-qPCR kits are costly, time-consuming, and shortages of supplies during the pandemics limit high-throughput testing requiring alternative solutions (Klein et al., 2020 ).


In our recent study (Dao Thi et al., 2020), we used reverse transcription loop-mediated isothermal amplification (RT-LAMP) (Notomi et al., 2020) as an alternative to detect SARS-CoV-2 RNA in clinical specimens. We developed and characterized colorimetric RT-LAMP assays on both purified and unpurified pharyngeal swab specimens. We also developed a multiplexed sequencing protocol based on tagmentation for enzymatic addition of barcoded sequencing library adapters. This enables the analysis of many RT-LAMP reactions at the same time. Here, we present detailed step-by-step protocols to further facilitate the application of RT-LAMP for mass testing.


Materials and Reagents

  1. 1.5 ml tubes (Eppendorf), room temperature

  2. Filter tips (for pipettes and liquidator), room temperature

  3. 96-well plate (Eppendorf, catalog number: 00 30128672 ), room temperature

  4. Nuclease-free water (Ambion, catalog number: AM9937 ), room temperature

  5. Ethanol for Molecular Biology

  6. WarmStart Colorimetric RT-LAMP 2× Master Mix (New England Biolabs, catalog number: M1800 ), -20 °C

  7. 10× primer mix for RT-LAMP assay as in Table 1 (Sigma-Aldrich), -20 °C


    Table 1. N gene primer for RT-LAMP assay. Primer sequences were designed by Zhang et al. (2020).

    Name Sequence Concentration in
    10× primer mix (µM)
    GeneN-A-F3 TGGCTACTACCGAAGAGCT 2
    GeneN-A-B3 TGCAGCATTGTTAGCAGGAT 2
    GeneN-A-FIP TCTGGCCCAGTTCCTAGGTAGTCCAGACGAATTCGTGGTGG 16
    GeneN-A-BIP AGACGGCATCATATGGGTTGCACGGGTGCCAATGTGATCT 16
    GeneN-A-LF GGACTGAGATCTTTCATTTTACCGT 4
    GeneN-A-LB ACTGAGGGAGCCTTGAATACA 4


  8. LAMP-sequencing primers as in Table 2 (Sigma-Aldrich), -20 °C


    Table 2. LAMP-sequencing primers. The full table is available as Table S4 in Dao Thi et al. (2020). [Phos] = phosphorylation, [SpcC3] = C3 spacer group, N, X, Y indicate one of the bases [GATC] (N are random bases while X and Y belong to respective inline barcodes used for multiplexing).

    Name Sequence
    Tn5hY-Rd2-Wat-SC3 [Phos]CTGTCTCTTATACACATCT[SpcC3]
    P5-UMI-xi5XXX-ME.fw CGGCGACCACCGAGATCTACACNNNNNNNNNXXXXXXXXXXXXCGTCGGCAGCG
    TCAGATGTGTATAAGAGACAG
    P5.fw AATGATACGGCGACCACCGAGATC
    P7nxt-GeneN-A-LBrc GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTGTATTCAAGGCTCCCTCAG
    T
    P7-xi7YYY CAAGCAGAAGACGGCATACGAGATYYYYYYYYYYYYTCTCGTGGGCTCGGAG


  9. Optically clear adhesive seal (Kisker Biotech, catalog number: GK480-OS ), room temperature

  10. Adhesive aluminum foil seal (Steinbrenner Laborsysteme, catalog number: SL-AM0550 ), room temperature

  11. Pierceable foil (Brooks Life Sciences, catalog number: 4ti-0566/96 ), room temperature

  12. 200 ng/µl Tn5 (E54K, L372P) Transposase (purified according to Hennig et al., 2018 , -80 °C)

  13. 0.2% SDS solution (room temperature)

  14. AMPureXP bead (Beckman Coulter, catalog number: A63881 ), 4 °C

  15. NEBNext Q5 HotStart polymerase (New England Biolabs, catalog number: M0543 ), -20 °C

  16. NucleoSpin Gel and PCR Clean-up mini kit (Macherey-Nagel, catalog number: 740609 ), room temperature

  17. NEBNext Library Quant Kit for Illumina (New England Biolabs, catalog number: E7630 ), -20 °C

  18. [Tris(hydroxymethyl)methylamino]propanesulfonic acid (TAPS)

  19. MgCl2

  20. Dimethylformamide (DMF)

  21. Freshly prepared 5× tagmentation buffer (see Recipes)

    Note: All chemicals purchased from Sigma-Aldrich except when indicated otherwise.

Equipment

  1. Pipetman L P2L, 0.2-2 μl (Gilead, catalog number: FA10001M )

  2. Pipetman L P20L, 2-20 μl (Gilead, catalog number: FA10003M )

  3. Pipetman L P200L, 20-200 μl (Gilead, catalog number: FA10005M )

  4. Pipetman L P1000L, 100-1,000 μl (Gilead, catalog number: FA10006M )

  5. Pipetman L Multichannel P8 x 20L, 2-20 μl (Gilead, catalog number: FA10009 )

  6. Liquidator 96 2-20 μl (Mettler Toledo, catalog number: LIQ-96-20 )

  7. Thermocycler (Biometra, TAdvanced 96 S )

  8. Absorbance reader (Tecan, model: Infinite M200/Spark Cyto )

  9. Centrifuge (Eppendorf, model: 5430 R )

  10. Table top centrifuge (Heraeus, model: Pico 21 )

  11. Magnetic stand (6 Tube Magnetic Stand; Ambion, catalog number: 10055 )

  12. NextSeq 550 machine (Illumina)

Procedure

A schematic diagram depicting the whole experimental procedure is shown in Figure 1.



Figure 1. Overview of experimental procedures. (a) RT-LAMP assays can be performed with purified or unpurified clinical specimens and then analyzed using a colorimetric or LAMP-sequencing read-out. (b) Flow-chart of LAMP-sequencing library preparation.


  1. RT-LAMP assays

      RT-LAMP assay on purified samples
    1. Isolate RNA from clinical specimen according to manufacturer’s protocol.

    2. Assemble RT-LAMP master mix in a 1.5 ml Eppendorf tube by adding 6.25 μl of the 2× Master Mix, 1.25 μl of 10× primer mix, and 4 μl nuclease-free water per reaction.

    3. Vortex and spin down.

    4. Distribute 11.5 μl of master mix into each well of a 96-well plate using a multichannel pipette.

    5. Add 1 μl of isolated RNA into wells with master mix.

    6. Seal plate with optically clear adhesive seal.

    7. Briefly spin down plate.

    8. Incubate for 30 min at 65 °C in a thermocycler (with the lid heated to 75 °C).


      RT-LAMP assay on unpurified samples
    1. For hot swab-to-RT-LAMP assays, pipette 50 μl of clinical specimen into 96-well plate and seal with pierceable foil.

    2. Heat up plate for 5 min at 95 °C in 96-well plate in a PCR cycler (with the lid heated to 105 °C).

    3. Cool down, spin briefly, and keep plate on ice.

    4. Assemble RT-LAMP master mix in a 1.5 ml Eppendorf tube by adding 10 μl of the 2× Master Mix, 2 μl of 10× primer mix, and 7 μl nuclease-free water per reaction.

    5. Vortex and spin down.

    6. Distribute 19 μl of master mix into each well of a 96-well plate using a multichannel pipette.

    7. For direct swab-to-RT-LAMP assays, pipette 1 μl of clinical specimen directly into wells with master mix.

    8. For hot assays, pipette 1 μl of prepared specimen (1-3) into wells with master mix.

    9. Seal plate with optically clear adhesive seal.

    10. Briefly spin down plate.

    11. Incubate for 30 min at 65 °C in a thermocycler (with the lid heated to 75 °C).


  2. RT-LAMP assay analysis

      Colorimetric read-out
    1. Cool down 96-well plate to 4 °C and spin down briefly.

    2. Place 96-well plate into absorbance reader.

    3. Measure absorbance at 434 nm and 560 nm.


      LAMP-sequencing

      (All concentrations are given as final concentrations in reactions.)

    1. Prepare transposon adapters by mixing individual barcoded adapter (P5-UMI-xi5XXX-ME.fw) with the primer Tn5hY-Rd2-Wat-SC3 at a final concentration of 25 μM per primer in 5 μM Tris-HCl (pH 8.0) in a 96-well PCR plate using the Liquidator. Heat up to 99 °C for 5 min and let the primers slowly anneal by cooling down to 20 °C within 15 min using a thermocycler.

    2. Mix transposase to a final concentration of 100 ng/µl with 1.25 μM annealed adapters from step 1 in 50 mM Tris-HCl (pH 7.5) in 96-well PCR plates using the Liquidator. Assemble transposons by incubating the reaction for 1 h at 23 °C in a thermocycler.

    3. Freshly prepare the 5× tagmentation buffer according to the indicated composition.

    4. Per well mix 1.2 μl of the RT-LAMP product (equivalent to ~200 ng DNA) with 1.5 μl of loaded transposase, 1.12 µl 5× tagmentation buffer from step 3 and 1.8 µl water to assemble the transposon reactions in 96-well PCR plates with the Liquidator. Incubate reactions at 55 °C for 10 min in a thermocycler.

    5. Stop the tagmentation reactions by adding 1.13 µl 0.2% SDS per well and incubate for 10 min at room temperature. Pool the reactions into one single reaction each plate.

    6. Perform size selection for fragments of approximately 300 to 600 bp by using the following two-step AMPure XP bead protocol (written for a pooled reaction from one plate).

      1. Mix 50 µl of pooled reaction with 50 µl of water.

      2. Remove large fragments by adding 55 µl of AMPure XP beads to the diluted samples. Mix by pipetting ten times and incubate at room temperature for 5 min. Separate beads from supernatant by placing on a magnetic stand for ~5 min. Transfer the supernatant to a fresh eppendorf tube using a pipette without transferring beads.

      3. Remove small fragments by adding 25 µl of fresh beads to the supernatant. Mix by pipetting ten times and incubate at room temperature for 5 min. Separate beads from supernatant by placing on a magnetic stand for ~5 min. Discard the supernatant containing the small fragments using a pipette without disturbing the bead pellet.

      4. Wash DNA bound to beads by two washes with ethanol. For this, add 200 µl ethanol (80%) to the beads, mix by pipetting ten times and incubate at room temperature for 5 min. Separate beads from ethanol by placing on a magnetic stand for ~5 min. Repeat this for a second wash. Let the beads air-dry for 10 min.

      5. Elute DNA from beads by adding 10 μl of 5 mM Tris-HCl (pH 8.5), incubating for 5 min at room temperature and separating on a magnetic rack for ~5 min.

    7. Perform one PCR reaction per plate using 1 µl of size-selected eluate from step 6 as a template. Prepare PCR reactions with RT-LAMP-specific and Tn5-adapter-specific primers (P7nxt-GeneN-A-LBrc and P7-xi7YYY, P5.fw) with the NEBNext Q5 HotStart polymerase according to the manufacturer’s instruction. Use the following PCR conditions for amplification with a thermocycler: Two cycles at 62 °C for annealing and 90 s elongation, followed by two cycles at 65 °C for annealing and 90 s elongation, and 13 cycles at 72 °C annealing and 90 s elongation.

    8. Pool all PCR reactions and perform a second size selection for fragments of approximately 400 to 550 bp: Run 20% of the pooled PCR reactions on a 2% agarose/Tris-acetate-EDTA gel, cut out the respective part of the lane and use a gel purification kit according to the manufacturer.

    9. Quantify the library using for example a qPCR-based library quantification kit.

    10. Perform a custom Illumina sequencing run on a NextSeq 550 machine based on the instructions of the manufacturer using 20% phiX spike-in and 136 cycles for the first read, 11 cycles to read the 11-nt-long plate index (i7) and 20 cycles to read the 11-nt-long well index (i5) and the 9-nt-long UMI.

Data analysis

  1. Colorimetric RT-LAMP analysis

    The results of the colorimetric RT-LAMP assay can be judged by naked eye. A clear color change from pink to orange or yellow is considered as SARS-CoV-2 positive after 30 min incubation at 65 °C. Color changes after 30 min can be caused by spurious amplification products and are therefore scored negative. For further validation, the RT-LAMP product can be analyzed by gel electrophoresis and should yield a distinct banding pattern as described previously (Dao Thi et al., 2020, see Figure 1 herein).

      When the assay is analyzed by a plate reader, subtract absorbance reads 560 nm from 434 nm (ΔOD). An ΔOD value > 0.3 is considered SARS-CoV-2 positive after 30 min incubation at 65 °C. For the hot swab-to-RT-LAMP assays, this read-out can be improved by subtracting the differences between the ΔOD values at time points 30 min and 10 min of the incubation at 65 °C.


  2. LAMP-sequencing analysis

    Raw NGS results (single-end fastq file) need first to be converted to count tables using a workflow, which can be downloaded from GitHub (https://github.com/anders-biostat/LAMP-Paper-Figures/tree/master/LAMP-sequencing_raw_read_processing). All the necessary software to run this workflow are summarized there. Individual processing steps can be run sequentially from inside this directory with the script ‘00-run_workflow.sh’ used for illustration the example file ‘LAMP-sequencing_raw_sample100k.fastq.gz’. In order to run the workflow with a different dataset one needs to adapt the pathnames in ‘00-run_workflow.sh’ accordingly. Two files are the result (‘counts.tsv’ and ‘counts.Rda’) which can be used for subsequent analysis. For example the count table to produce the respective figures for our RT-LAMP study (Dao Thi et al., 2020) is also present in this GitHub repository.

Notes

All work with crude SARS-CoV-2 clinical specimens should be carried out in a biosafety level 2 cabinet until inactivation. We found that both purified and unpurified pharyngeal swab specimens as well as saliva specimens were compatible with RT-LAMP assays. Other types of specimens have to be tested.  

  In order to avoid contaminations and RNA degradation, all steps are carried out using filter tips and wearing gloves. In addition, keep clinical specimens on ice as much as possible to prevent RNA degradation. Master mix and test samples should be pipetted at different workplaces using different sets of pipettes. Ideally, the person executing the protocols has experience in molecular biology. Additional important considerations when using RT-LAMP reagents are listed in the Supplementary Material of our previously published work (Dao Thi et al., 2020).

Recipes

  1. 5× tagmentation buffer

    (Always prepare fresh.)

    1 vol of 10× TAPS buffer (100 mM [Tris(hydroxymethyl)methylamino]propanesulfonic acid (TAPS) (pH 8.5), 50 mM MgCl2)

    1 vol of 100% (v/v) dimethylformamide (DMF)

Acknowledgments

This protocol was modified from our original method published previously (Dao Thi et al., 2020). K.H. was supported through a grant by the Deutsche Forschungsgemeinschaft (DFG; grant no. KN498/11-1) to M.K. V.L.D.T., was supported by the Chica and Heinz Schaller foundation. We would like to acknowledge the EMBL Protein Expression and Purification facility for production of the Tn5 enzyme.

Competing interests

The authors declare no competing interests.

References

  1. Corman, V. M., Landt, O., Kaiser, M., Molenkamp, R., Meijer, A., Chu, D. K., Bleicker, T., Brunink, S., Schneider, J., Schmidt, M. L., Mulders, D. G., Haagmans, B. L., van der Veer, B., van den Brink, S., Wijsman, L., Goderski, G., Romette, J. L., Ellis, J., Zambon, M., Peiris, M., Goossens, H., Reusken, C., Koopmans, M. P. and Drosten, C. (2020). Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR. Euro Surveill 25(3): 2000045.
  2. Dao Thi, V. L., Herbst, K., Boerner, K., Meurer, M., Kremer, L. P., Kirrmaier, D., Freistaedter, A., Papagiannidis, D., Galmozzi, C., Stanifer, M. L., Boulant, S., Klein, S., Chlanda, P., Khalid, D., Barreto Miranda, I., Schnitzler, P., Krausslich, H. G., Knop, M. and Anders, S. (2020). A colorimetric RT-LAMP assay and LAMP-sequencing for detecting SARS-CoV-2 RNA in clinical samples. Sci Transl Med 12(556): eabc7075.
  3. Hennig, B. P., Velten, L., Racke, I., Tu, C. S., Thoms, M., Rybin, V., Besir, H., Remans, K. and Steinmetz, L. M. (2018). Large-Scale Low-Cost NGS Library Preparation Using a Robust Tn5 Purification and Tagmentation Protocol. G3 (Bethesda) 8(1): 79-89.
  4. Klein, S., Müller, T. G., Khalid, D., Sonntag-Buck, V., Heuser, A. M., Glass, B., Meurer, M., Morales, I., Schillak, A., Freistaedter, A., Ambiel, I., Winter, S. L., Zimmermann, L., Naumoska, T., Bubeck, F., Kirrmaier, D., Ullrich, S., Barreto Miranda, I., Anders, S., Grimm, D., Schnitzler, P., Knop, M., Kräusslich, H. G., Dao Thi, V. L., Börner, K. and Chlanda, P. (2020). SARS-CoV-2 RNA Extraction Using Magnetic Beads for Rapid Large-Scale Testing by RT-qPCR and RT-LAMP. Viruses 12(8): 863.
  5. Li, H., Liu, L., Zhang, D., Xu, J., Dai, H., Tang, N., Su, X. and Cao, B. (2020). SARS-CoV-2 and viral sepsis: observations and hypotheses. Lancet 395(10235): 1517-1520.
  6. Notomi, T., Okayama, H., Masubuchi, H., Yonekawa, T., Watanabe, K., Amino, N. and Hase, T. (2000). Loop-mediated isothermal amplification of DNA. Nucleic Acids Res 28(12): e63.
  7. Zhang, Y. , Odiwuor, N., Xiong, J., Sun, L., Nyaruaba, R. O., Wei, H. and Tanner, N. A. (2020). Rapid molecular detection of SARS-CoV-2 (COVID-19) virus RNA using colorimetric LAMP. medRxiv. https://doi.org/10.1101/2020.02.26.20028373.

简介

[摘要]在大流行期间(例如由SARS-CoV-2冠状病毒引起的大流行),需要一种简单的方法来快速测试大量人员。作为通过常规qPCR检测病毒RNA的一种更快且资源更少的替代方法,我们使用了逆转录环介导的等温扩增(RT-LAMP)。我们先前在纯化和未纯化的SARS-CoV-2临床标本上建立了比色RT-LAMP分析方法,并进一步开发了多重测序方案(LAMP测序)来同时分析许多RT-LAMP反应的结果(Dao Thi等)等人,2020年)。在此工作的基础上,我们在此提供针对RT-LAMP分析和读数的分步操作规程。

[背景]新的SARS-CoV-2冠状病毒构成了重大的公共卫生问题(Li等人,2020年综述)。在缺乏有效的抗病毒治疗和保护性疫苗的情况下,通过大量检测防止局部暴发至关重要。检测SARS-CoV-2感染的标准诊断流程基于以下条件:从临床标本中分离病毒RNA,将RNA转录为cDNA的逆转录(RT)反应以及通过半定量DNA聚合酶链反应进行检测(qPCR)(Corman等,2020)。然而,商业化的RNA分离和RT-qPCR试剂盒价格昂贵,耗时且大流行期间供应短缺,从而限制了高通量测试,需要其他解决方案(Klein等人,2020)。

在我们最近的研究中(Dao Thi等人,2020),我们使用逆转录环介导的等温扩增(RT-LAMP)(Notomi等人,2020)作为在临床标本中检测SARS-CoV-2 RNA的替代方法。我们在纯化和未纯化的咽拭子标本上开发并表征了比色RT-LAMP测定法。我们还开发了基于标签的多重测序方案,用于酶促添加条形码测序文库衔接子。这样可以同时分析许多RT-LAMP反应。在这里,我们提出了详细的分步协议,以进一步促进RT-LAMP在大规模测试中的应用。

关键字:逆转录环介导等温扩增, 环介导等温扩增测序, SARS-CoV-2检测, Tn5测序建库方法, 比色测定



材料和试剂

1.5 ml管(Eppendorf),室温
过滤嘴(用于移液器和清液器),室温
96孔板(Eppendorf,目录号:0030128672),室温
无核酸酶的水(Ambion,目录号:AM9937),室温
分子生物学用乙醇
WarmStart比色RT-LAMP 2 ×预混液(New England Biolabs,目录号:M1800),-20°C
如表1所示的用于RT-LAMP分析的10 ×引物混合物(Sigma-Aldrich),-20°C

表1.用于RT-LAMP测定的N基因引物。引物序列由Zhang等人设计。(2020)。


姓名


顺序


集中在


10 ×底漆混合物(µM)


基因N-A-F3


TGGCTACTACCGAAGAGCT


2个


基因N-A-B3


TGCAGCATTGTTAGCAGGAT


2个


基因N-A-FIP


TCTGGCCCAGTTCCTAGGTAGTCCAGACGAATTCGTGGTGG


16


基因N-A-BIP


AGACGGCATCATATGGGTTGCACGGGTGCCAATGTGATCT


16


基因N-A-LF


GGACTGAGATCTCTTTCATTTTACCGT


4


基因N-A-LB


ACTGAGGGAGCCTTGAATACA


4

表2中的LAMP测序引物(Sigma-Aldrich),-20°C

表2. LAMP测序引物。完整的表可作为Dao Thi等人的表S4获得。(2020)。[Phos] =磷酸化,[SpcC3] = C3间隔基,N,X,Y表示碱基之一[GATC](N是随机碱基,而X和Y分别属于用于复用的内联条形码)。


姓名


顺序


Tn5hY-Rd2-Wat-SC3


[Phos] CTGTCTCTTATACACATCT [SpcC3]


P5-UMI-xi5XXX-ME.fw


CGGCGACCACCGAGATCTACACNNNNNNNNNXXXXXXXXXXXXCGTCGGCAGCGTCAGATGTGTATAAGAGACAG


P5


AATGATACGGCGACCACCGAGATC


P7nxt-GeneN-A-LBrc


GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTGTATTCAAGGCTCCCTCAGAGT


P7-xi7YYY


CAAGCAGAAGACGGCATACGAGATYYYYYYYYYYYYTCTCGTGGGCTCGGAG

光学透明的密封胶(Kisker Biotech,目录号:GK480-OS),室温
铝箔胶密封(Steinbrenner Laborsysteme,目录号:SL-AM0550),室温
可刺穿的铝箔(布鲁克生命科学,目录号:4ti-0566 / 96),室温
200 ng / µl Tn5 (E54K,L372P)转座酶(根据Hennig等人的方法纯化,2018,-80°C)
0.2%SDS溶液(室温)
AMPureXP磁珠(贝克曼库尔特(Beckman Coulter),目录号:A638 81),4°C
NEBNext Q5 HotStart聚合酶(New England Biolabs,目录号:M0543),-20°C
核苷销凝胶和PCR净化-向上mini试剂盒(马歇雷-Nagel的,目录号:740609),室温下
NEBNext用于Illumina的文库定量试剂盒(New England Biolabs,目录号:E7630),-20°C
[三(羟甲基)甲基氨基]丙烷磺酸(TAPS)
氯化镁2
二甲基甲酰胺(DMF)
新鲜制备的5 ×标记缓冲液(请参见食谱)
注意:所有从Sigma-Aldrich购买的化学品,除非另有说明。

设备

Pipetman L P2L,0.2-2μl (Gilead,目录号:FA10001M)
移液器L P20L,2-20μl(Gilead,目录号:FA10003M)
移液器L P200L,20-200μl(Gilead,目录号:FA10005M)
移液器大号P1000L,100-1 ,000微升(Gilead公司,目录号:FA10006M)
Pipetman L多通道P8 x 20L,2-20μl (Gilead,目录号:FA10009)
清算96 2-20 μ升(Mettler Toledo的,目录号:LIQ-96-20)
Thermocycle r(Biometra,TAdvanced 96 S)
吸光度读取器(Tec an,型号:Infinite M200 / Spark Cyto)
离心机(Eppendorf,型号:5430 R)
台式离心机(Heraeus,型号:Pico 21)
磁力架(6管磁力架; Ambion,目录号:10055)
NextSeq 550机器(Illumina)

程序

图1中显示了描述整个实验过程的示意图。





图1.实验程序概述。(a)可以对纯化或未纯化的临床标本进行RT-LAMP测定,然后使用比色法或LAMP测序读数进行分析。(b)LAMP测序文库制备流程图。

RT-LAMP分析
纯化样品的RT-LAMP测定


根据制造商的协议从临床样本中分离RNA。
通过加入6.25装配在1.5ml Eppendorf管RT-LAMP主混合物μ 2的升×主混合物,1.25 μ 10升×引物混合物,和4 μ每个反应升无核酸酶水。
涡旋并旋转下来。
分发11.5 μ主混合物的升成的96孔每孔用多通道移液器板上。
加入1 μ分离的RNA的升与主混合物井。
用光学透明的粘合剂密封的密封板。
短暂地旋转板。
在热循环仪(盖加热到75°C)中于65°C孵育30分钟。

未纯化样品的RT-LAMP测定


对于热拭子至RT-LAMP分析,将50μl临床样品吸移到96孔板中,并用可刺穿的箔密封。
在PCR循环仪中将板在96孔板中于95°C加热5分钟(盖子加热至105°C)。
冷却,短暂旋转,然后将其放在冰上。
通过加入10装配在1.5ml Eppendorf管RT-LAMP主混合物μ 2的升×主混合物,2 μ的10升×引物混合物,和7 μ每个反应升无核酸酶水。
涡旋并旋转下来。
分发19 μ主混合物的升成的96孔每孔用多通道移液器板。
对于直接拭子到RT-LAMP分析,将1μl临床样品直接用预混液吸进孔中。
对于热测定,将1μl准备好的标本(1-3)移入带有预混液的孔中。
用光学透明的粘合剂密封的密封板。
短暂地旋转板。
在热循环仪中(盖加热到75°C)在65°C下孵育30分钟。

RT-LAMP分析
比色读数


将96孔板冷却至4°C,然后短暂旋转。
将96孔板放入吸光度读数器中。
测量在434 nm和560 nm处的吸光度。

LAMP测序


(所有浓度均以反应中的最终浓度给出。)


通过将单个带条形码的适配器(P5-UMI-xi5XXX-ME.fw)与引物Tn5hY-Rd2-Wat-SC3混合,以每个引物的终浓度25μM在5μMTris-HCl(pH 8 .0 )中制备转座子适配器使用Liquidator在96孔PCR板中扩增。加热至99 °C,持续5分钟,然后使用热循环仪在15分钟内将其冷却至20°C,以使引物缓慢退火。
使用Liquidator在96孔PCR板中的50 mM Tris-HCl(pH 7.5)中,将步骤1中的1.25μM退火接头将转座酶混合至终浓度为100 ng /μl。通过在23°C的热循环仪中将反应孵育1小时来组装转座子。
根据指示的成分新鲜制备5 ×标记缓冲液。
每孔英里×1.2与1.5微升加载的转座,1.12微升5微升RT-LAMP产物(相当于〜200纳克DNA)的× TA gmentation从步骤3和1.8μ缓冲升水组装在96转座子反应带有Liquidator的PCR板。在55°C的热循环仪中将反应孵育10分钟。
通过每孔添加1.13 µl 0.2%SDS终止标记反应,并在室温下孵育10分钟。每个板将反应合并为一个单一反应。
通过使用以下两步AMPure XP珠操作规程(针对从一个板中合并的反应编写),对大约300到600 bp的片段进行大小选择。
将50 µl合并的反应与50 µl的水混合。
通过将55 µl AMPure XP珠粒添加到稀释的样品中来除去较大的碎片。通过移液十次混合,并在室温下孵育5分钟。通过将其放在磁力架上约5分钟,将珠子与上清液分开。使用移液器将上清液转移到新的eppendorf管中,而不转移珠子。
通过向上清液中加入25 µl新鲜的珠子来去除小碎片。通过移液十次混合,并在室温下孵育5分钟。通过将其放在磁力架上约5分钟,将珠子与上清液分开。使用移液器丢弃含有小碎片的上清液,而不会干扰珠粒。
用乙醇洗涤两次,将与小珠结合的DNA洗净。为此,将200 µl乙醇(80%)添加到珠子中,通过移液十次进行混合,然后在室温下孵育5分钟。通过将其放在磁力架上约5分钟,将珠子与乙醇分开。重复此步骤进行第二次清洗。让珠子风干10分钟。
加入10μl5 mM Tris-HCl(pH 8.5),从珠子上洗脱DNA,在室温下孵育5分钟,并在磁力架上分离约5分钟。
使用来自步骤6的1 µl大小选择的洗脱液作为模板,每块板进行一个PCR反应。根据制造商的说明,使用NEBNext Q5 HotStart聚合酶用RT-LAMP特异性和Tn5-adapter特异性引物(P7nxt-GeneN-A-LBrc和P7-xi7YYY,P5.fw)准备PCR反应。使用以下PCR条件通过热循环仪进行扩增:在62°C进行两个循环进行退火和90 s延长,然后在65°C进行两个循环进行退火和90 s延长,然后在72°C进行90 s退火进行13个循环。伸长。
合并所有PCR反应,并对大约400至550 bp的片段进行第二次大小选择:在2%琼脂糖/ Tris-醋酸盐-EDTA凝胶上运行20%的PCR反应,切下泳道的相应部分并使用根据制造商的凝胶纯化套件。
使用例如基于qPCR的文库定量试剂盒对文库进行定量。
根据制造商的说明,在NextSeq 550机器上执行自定义Illumina测序,使用20%phiX尖峰和136个循环进行首次读取,使用11个循环读取11 nt长的板索引(i7)和20循环读取11 nt长的阱指数(i5)和9 nt长的UMI。

数据分析

比色RT-LAMP分析
比色RT-LAMP测定的结果可以用肉眼判断。在65°C下孵育30分钟后,从粉红色变为橙色或黄色的明显颜色变化被认为是SARS-CoV-2阳性 ℃。30分钟后的颜色变化可能是由虚假的扩增产物引起的,因此被评为阴性。为了进一步验证,RT-LAMP产物可通过凝胶电泳进行分析,并应产生如先前所述的独特的条带模式(Dao Thi等人,2020,请参见此处的图1)。


用酶标仪分析测定值时,从434 nm(ΔOD)中减去560 nm的吸光度。在65 °C孵育30分钟后,将ΔOD值> 0.3视为SARS-CoV-2阳性。对于热拭子到RT-LAMP的检测,可以通过减去在65 °C下孵育30分钟和10分钟的时间点ΔOD值之间的差异来改善此读数。


           

LAMP测序分析
首先需要使用工作流将原始NGS结果(单端fastq文件)转换为计数表,该工作流可从GitHub(https://github.com/anders-biostat/LAMP-Paper-Figures/tree/master下载)/ LAMP-sequencing_raw_read_processing)。此处总结了运行此工作流程的所有必需软件。各个处理步骤可以顺序地从该目录内的脚本00 -run_workflow.sh”运行我们编的用于说明的示例文件‘LAMP-sequencing_raw_sample100k.fastq.gz’。为了使用不同的数据集运行工作流,需要相应地修改“ 00-run_workflow.sh”中的路径名。结果为两个文件(“ counts.tsv”和“ counts.Rda”),可用于后续分析。例如,此GitHub存储库中还提供了用于为我们的RT-LAMP研究生成相应数字的计数表(Dao Thi等人,2020年)。

笔记

应对SARS-CoV-2原始临床标本进行的所有工作均应在生物安全等级为2的机柜中进行,直至灭活。我们发现,纯化和未纯化的咽拭子标本以及唾液标本均与RT-LAMP分析兼容。其他类型的标本也必须进行测试。


为了避免污染和RNA降解,所有步骤均使用滤嘴和戴手套进行。另外,尽可能将临床标本放在冰上以防止RNA降解。应当在不同的工作场所使用不同的移液器吸取预混液和测试样品。理想情况下,执行方案的人员应具有分子生物学经验。使用RT-LAMP试剂时的其他重要注意事项在我们先前发表的工作的补充材料中列出(Dao Thi等人,2020年)。

菜谱

5 ×标签缓冲
(总是准备新鲜。)


1体积的10 × TAPS缓冲液(100 mM [三(羟甲基)甲基氨基]丙烷磺酸(TAPS)(pH 8.5),50 mM MgCl 2 )


1体积的100%(v / v)二甲基甲酰胺(DMF)

致谢

该协议是根据我们先前发布的原始方法(Dao Thi等人,2020)修改而来的。KH由Deutsche Forschungsgemeinschaft(DFG;授权号KN498 / 11-1)对MKVLDT的资助提供支持,并得到Chica和Heinz Schaller基金会的支持。我们要感谢用于生产Tn5酶的EMBL蛋白表达和纯化工具。

利益争夺

作者宣称没有利益冲突。

参考

1. Corman,VM,Landt,O.,Kaiser,M.,Molenkamp,R.,Meijer,A.,Chu,DK,Bleicker,T.,Brunink,S.,Schneider,J.,Schmidt,ML,Mulders ,DG,Haagmans,BL,van der Veer,B.,van den Brink,S.,Wijsman,L.,Goderski,G.,Romette,JL,Ellis,J.,Zambon,M.,Peiris,M。 Goossens,H.,Reusken,C.,Koopmans,MP和Drosten,C.(2020年)。通过实时RT-PCR检测2019年新型冠状病毒(2019-nCoV)。欧洲监测25(3):2000045。     

2. Dao Thi,VL,Herbst,K.,Boerner,K.,Meurer,M.,Kremer,LP,Kirrmaier,D.,Freistaedter,A.,Papagiannidis,D.,Galmozzi,C.,Stanifer,ML, Boulant,S.,Klein,S.,Chlanda,P.,Khalid,D.,Barreto Miranda,I.,Schnitzler,P.,Krausslich,HG,Knop,M.和Anders,S.(2020年)。比色RT-LAMP测定和LAMP测序用于检测临床样品中的SARS-CoV-2 RNA。Sci Transl Med 12(556):eabc7075。     

3. Hennig,BP,Velten,L.,Racke,I.,Tu,CS,Thoms,M.,Rybin,V.,Besir,H.,Remans,K. and Steinmetz,LM(2018)。使用稳健的Tn5纯化和标记方案进行大规模低成本NGS库制备。G3(贝塞斯达)8(1):79-89。                   

4. Klein,S.,Müller,TG,Khalid,D.,Sonntag-Buck,V.,Heuser,AM,Glass,B.,Meurer,M.,Morales,I.,Schillak,A.,Freistaedter,A 。,Ambiel,I.,Winter,SL,Zimmermann,L.,Naumoska,T.,Bubeck,F.,Kirrmaier,D.,Ullrich,S.,Barreto Miranda,I.,Anders,S.,Grimm,D 。,Schnitzler,P.,Knop,M.,Kräusslich,HG,Dao Thi,VL,Börner,K.和Chlanda,P.(2020)。使用磁珠提取SARS-CoV-2 RNA,通过RT-qPCR和RT-LAMP快速进行大规模测试。病毒12(8):863。     

5. Li H,Liu,L.,Zhang D.,Xu,J.,Dai H.,Tang N.,Su,X. and Cao,B.(2020)。SARS-CoV-2和病毒性败血症:观察和假设。柳叶刀395(10235):1517-1520。     

6. T. Notomi,H。冈山,H.Masubuchi,H.Yonekawa,K。渡边,K.,Amino,N。Hase(2000)。DNA的环介导等温扩增。Nucleic Acids Res 28(12):e63。     

7. Zhang,Y.,Odiwuor,N.,熊J.,Sun L.,Nyaruaba,RO,Wei H.和Tanner,NA(2020)。使用比色LAMP快速检测SARS-CoV-2(COVID-19)病毒RNA的分子。medRxiv 。https://doi.org/10.1101/2020.02.26.20028373。      
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Copyright: © 2021 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Herbst, K., Meurer, M., Kirrmaier, D., Anders, S., Knop, M. and Dao Thi, V. L. (2021). Colorimetric RT-LAMP and LAMP-sequencing for Detecting SARS-CoV-2 RNA in Clinical Samples. Bio-protocol 11(6): e3964. DOI: 10.21769/BioProtoc.3964.
  2. Dao Thi, V. L., Herbst, K., Boerner, K., Meurer, M., Kremer, L. P., Kirrmaier, D., Freistaedter, A., Papagiannidis, D., Galmozzi, C., Stanifer, M. L., Boulant, S., Klein, S., Chlanda, P., Khalid, D., Barreto Miranda, I., Schnitzler, P., Krausslich, H. G., Knop, M. and Anders, S. (2020). A colorimetric RT-LAMP assay and LAMP-sequencing for detecting SARS-CoV-2 RNA in clinical samples. Sci Transl Med 12(556): eabc7075.
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