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
发布: 2021年03月20日第11卷第6期 DOI: 10.21769/BioProtoc.3964 浏览次数: 5531
评审: Kenji SugiyamaNathan Rhys JamesSonali ChaturvediAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Aug 2020
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
1.5 ml tubes (Eppendorf), room temperature
Filter tips (for pipettes and liquidator), room temperature
96-well plate (Eppendorf, catalog number: 00 30128672 ), room temperature
Nuclease-free water (Ambion, catalog number: AM9937 ), room temperature
Ethanol for Molecular Biology
WarmStart Colorimetric RT-LAMP 2× Master Mix (New England Biolabs, catalog number: M1800 ), -20 °C
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 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
TCAGATGTGTATAAGAGACAGP5.fw AATGATACGGCGACCACCGAGATC P7nxt-GeneN-A-LBrc GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTGTATTCAAGGCTCCCTCAG
TP7-xi7YYY CAAGCAGAAGACGGCATACGAGATYYYYYYYYYYYYTCTCGTGGGCTCGGAG Optically clear adhesive seal (Kisker Biotech, catalog number: GK480-OS ), room temperature
Adhesive aluminum foil seal (Steinbrenner Laborsysteme, catalog number: SL-AM0550 ), room temperature
Pierceable foil (Brooks Life Sciences, catalog number: 4ti-0566/96 ), room temperature
200 ng/µl Tn5 (E54K, L372P) Transposase (purified according to Hennig et al., 2018 , -80 °C)
0.2% SDS solution (room temperature)
AMPureXP bead (Beckman Coulter, catalog number: A63881 ), 4 °C
NEBNext Q5 HotStart polymerase (New England Biolabs, catalog number: M0543 ), -20 °C
NucleoSpin Gel and PCR Clean-up mini kit (Macherey-Nagel, catalog number: 740609 ), room temperature
NEBNext Library Quant Kit for Illumina (New England Biolabs, catalog number: E7630 ), -20 °C
[Tris(hydroxymethyl)methylamino]propanesulfonic acid (TAPS)
MgCl2
Dimethylformamide (DMF)
Freshly prepared 5× tagmentation buffer (see Recipes)
Note: All chemicals purchased from Sigma-Aldrich except when indicated otherwise.
Equipment
Pipetman L P2L, 0.2-2 μl (Gilead, catalog number: FA10001M )
Pipetman L P20L, 2-20 μl (Gilead, catalog number: FA10003M )
Pipetman L P200L, 20-200 μl (Gilead, catalog number: FA10005M )
Pipetman L P1000L, 100-1，000 μl (Gilead, catalog number: FA10006M )
Pipetman L Multichannel P8 x 20L, 2-20 μl (Gilead, catalog number: FA10009 )
Liquidator 96 2-20 μl (Mettler Toledo, catalog number: LIQ-96-20 )
Thermocycler (Biometra, TAdvanced 96 S )
Absorbance reader (Tecan, model: Infinite M200/Spark Cyto )
Centrifuge (Eppendorf, model: 5430 R )
Table top centrifuge (Heraeus, model: Pico 21 )
Magnetic stand (6 Tube Magnetic Stand; Ambion, catalog number: 10055 )
NextSeq 550 machine (Illumina)
Procedure
文章信息
版权信息
© 2021 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
- 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.
- 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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分类
分子生物学 > RNA > RNA 检测
微生物学 > 病原体检测 > RT-LAMP
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