Colorimetric RT-LAMP and LAMP-sequencing for Detecting SARS-CoV-2 RNA in Clinical Samples

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