1 LAMP protocols for detection of SARS-CoV-2 genomic RNA _(Pr O.

De Backer, UNamur)
2
3 1. Equipment
4
5 - Vortex apparatus (only for RNA extraction).
6 - high speed 1,5ml tube centrifuge (only for RNA extraction).
7 - thermostatic oven (preferred) or water bath or dry bath.
8
9 2. Vessels
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11 - 1.5 tubes for RNA extraction
12 96 well sealable microwell plates and sealing tape
13 (alternatively: 1.5ml or 0.5 ml or 0, ml tubes) for LAMP.
14 NB : microwell plates are preferred to tubes because :
15 o easy management of a large number of samples.
16 o easy record of the visual inspection readout (wells are numbered and a single picture of
17 the plate)
18 o more robust results (probably because of less evaporation)
19
20 3. Reagents for RNA extraction
21 - high speed 1,5ml tube centrifuge
22 - Chloroform (Sigma-Aldrich)
23 - GlycoBlue (Thermofisher Scientifc) (or Glycogen or yeast tRNA or any other RNase free ballast for
24 ethanol co-precipitation to increases visibility of the RNA pellet)
25 - Isopropanol (Sigma-Aldrich)
26 - Ethanol 75% (Sigma-Aldrich). In a falcon 50ml tube, add 12,5 ml of RNAse free water to 37,5ml of
27 ethanol 96-100% (Sigma-Aldrich).
28 - RNAse free water
29
30 4. Reagents for Colorimetric LAMP
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32 - WarmStart® Colorimetric LAMP 2X Master Mix New England Biolabs cat # M1800
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34 (alternatively, could be homemade with Bst 3.0 DNA Polymerase, New England Biolabs cat#M0374 :
35 see Visual detection of isothermal nucleic acid ampli cation using pH-sensitive dyes
36 Nathan A. Tanner, Yinhua Zhang, and Thomas C. Evans Jr.
37 DNA Enzymes Division, New England Biolabs, Ipswich, MA BioTechniques 58:59-68 (February 2015)
38 doi 10.2144/000114253)
39
40 - Two mix of 6 primers (synthetic oligonucleotides) each (10 x)
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42 Primers should be HPLC purified (at least FIP and BIP primers)
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44 “GeneN_B” set of primers
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46 GeneN-B-F3 5’ ACCGAAGAGCTACCAGACG
47 GeneN-B-B3 5’ TGCAGCATTGTTAGCAGGAT
48 GeneN-B-FIP 5’ TCTGGCCCAGTTCCTAGGTAGTTCGTGGTGGTGACGGTAA
49 GeneN-B-BIP 5’ AGACGGCATCATATGGGTTGCACGGGTGCCAATGTGATCT
50 GeneN-B-LF 5’ CCATCTTGGACTGAGATCTTTCATT
51 GeneN-B-LB 5’ ACTGAGGGAGCCTTGAATACA
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53
54 “GeneN_ID21” set of primers
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56 F3_ID21 5’ GCCAAAAGGCTTCTACGCA
57 B3_ID21 5’ TTGCTCTCAAGCTGGTTCAA
58 FIP_ID21 5’ TCCCCTACTGCTGCCTGGAGGCAGTCAAGCCTCTTCTCG
59 BIP_ID21 5’ TCTCCTGCTAGAATGGCTGGCATCTGTCAAGCAGCAGCAAAG
60 LF_ID21 5’ TGTTGCGACTACGTGATGAGGA
61 LB _ID21 5’ ATGGCGGTGATGCTGCTCT
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63 Prepare the two 10X LAMP Primer Mix (one with primers « GeneN-B », the other with primers
64 « ID21 »)
65
66 10X LAMP Primer Mix
67 FIP 16 μM (1x final concentration in LAMP reaction = 1.6 μM)
68 BIP 16 μM (1x final concentration in LAMP reaction = 1.6 μM)
69 F3 2 μM (1x final concentration in LAMP reaction = 0.2 μM)
70 B3 2 μM (1x final concentration in LAMP reaction = 0.2 μM)
71 LF 4 μM (1x final concentration in LAMP reaction = 0.4 μM)
72 LB 4 μM (1x final concentration in LAMP reaction = 0.4 μM)
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 75 5. Inactivation of viral infectivity and RNA extraction (other RNA extraction protocols can be
76 used)
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78 This step and the preparation of LAMP amplification should be performed in a “preLAMP” area to
79 avoid contamination.
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81 RNA is extracted from 100 of μl of swab transport medium using TRI Reagent from Sigma-Aldrich or
82 TRIzol from Invitrogen or QIAzol from QIAGEN and dissolved in 30 μl of RNase free water. Store at -
83 20°C (or -80°C if available).
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85 Clinical specimen processing will be performed in a class II biological safety cabinet by experienced
86 researcher wearing appropriate protective equipment (gowns and gloves).
87 From now, the whole process is carried out using filtered tips.
88
89 - Spin the collection tube 200 g, 1min, to make sure the sample is at the bottom of the tube
90 Use a centrifuge with sealed buckets
91 - Clean the centrifuge
92 - Transfer 100 µl fraction of the clinical sample in a 1,5 ml tube containing 1.0 ml of TRI Reagent.
93 - mix gently by inverting the tube. This procedure inactivates sample upon arrival in TRI Reagent.
94 - incubate 5 min at room temperature. If necessary, inactivated samples can be stored at -20 °C (or
95 -80 °C) for further processing.
96 - Add 200 µl of chloroform and vortex for 10 sec
97 - Incubate for 5 min at room temperature
98 - Centrifuge at 12,000 g for 10 min preferentially at 4 °C
99 - Transfer 500 µl of the colorless upper aqueous phase (containing the RNA) in 1.5 ml tube
100 containing 3 µl of Glycoblue (co-precipitant to increases visibility of the RNA pellet)
101 - importantly avoid any contact with the ring or the lower organic phase (pink)
102 Important note: the organic and aqueous phase can be inverted, i.e. the organic pink phase
103 can be above the clear aqueous phase. In this case, add 100ul of RNAse free water to the
104 sample, vortex and centrifuge at 12,000 g for 10 min at 4 °C.
105 - Add 500 µl of isopropanol and vortex for 3 sec
106 - Incubate for 5 min at room temperature
107 - Centrifuge at 12,000 to 14,000 g for 10 min
108 - The RNA pellet forms a blue pellet on the bottom of the tube
109 - Discard the supernatant
110 - Add 900 µl of 75 % ethanol
111 - Gently mix by inverting the tubes (avoid to detach the pellet)
112 - Centrifuge for 5 min at 12,000 to 14,000 g at 4 °C
113 - Aspirate slowly the supernatant with a 1 ml pipette by avoiding contact with the blue pellet.
114 - Centifuge briefly again.
115 - Use a narrow tip to remove residual ethanol
116 - To dry the pellet, leave the tube open under the chemical hood until complete ethanol
117 evaporation. This step lasts about 5 min. Do not over dry the RNA by letting the sample dry more
118 than 10 min.
119 - Dissolve the pellet in 30 µl of RNAse free water
120 - Incubate at room temperature until complete dissolution of the blue pellet
121 - If necessary, RNA can be stored at -80C for further processing
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123
124 6. Colorimetric LAMP protocol
125
126 Prepare on ice (if available) a mixture (scale up) for the desired number of samples plus positive
127 (positive RNA or synthetic DNA or RNA) and negative controls (negative sample and H2O).
128
129 Per sample :
130 Colorimetric LAMP 2X Master Mix 12.5 μl
131 (must be pink when thawed)
132 10 X Primer Mix GeneN_B 2.5 μl
133 10 X Primer Mix GeneN_ID21 2.5 μl
134 H2O 3.5 μl
135
136 - Dispense 21 μl in wells (or tubes)
137 - Put on ice (if available)
138 - Add 4 μl of RNA sample, mix carefully by up and down pipetting
139
140 - Seal the plate with plastic film
141
142 - Incubate at 65°C for 35 minutes.
143
144 We incubate the plate in a home-made small sandbox placed in an air incubator. Check that the sand
145 is at 65°C.
146

147
148
149 - Inspect visually the plate.
150
151 Positive controls should be yellow, negative controls should be pink.
152 Orange is also to be considered as positive.
153 - Take a picture of the plate upside down.
154 See below for an example.
155
156 The plate can be frozen and snapped later.
157 Do not open the plate to avoid contamination.
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166
167 Electrophoresis readout (optional)
168
169 Run 15 μl of the reaction mixture on a 0,7% agarose gel containing ethidium bromide.
170 Positive sample should display amplification products including discrete bands corresponding to the
171 “amplicon” and concatemeres of the amplicon (see picture).
172

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176 Gene N showing the location of F3 (sense, left) and B3 (antisense, right) primers
177 as follows:
178
179 Gene N-B
180 ID21
181
182 ATGTCTGATAATGGACCCCAAAATCAGCGAAATGCACCCCGCATTACGTTTGGTGGACCCTCAGATTCAACTGG
183 CAGTAACCAGAATGGAGAACGCAGTGGGGCGCGATCAAAACAACGTCGGCCCCAAGGTTTACCCAATAATAC
184 TGCGTCTTGGTTCACCGCTCTCACTCAACATGGCAAGGAAGACCTTAAATTCCCTCGAGGACAAGGCGTTCCAA
185 TTAACACCAATAGCAGTCCAGATGACCAAATTGGCTACTACCGAAGAGCTACCAGACGAATTCGTGGTGGTGA
186 CGGTAAAATGAAAGATCTCAGTCCAAGATGGTATTTCTACTACCTAGGAACTGGGCCAGAAGCTGGACTTCCC
187 TATGGTGCTAACAAAGACGGCATCATATGGGTTGCAACTGAGGGAGCCTTGAATACACCAAAAGATCACATTG
188 GCACCCGCAATCCTGCTAACAATGCTGCAATCGTGCTACAACTTCCTCAAGGAACAACATTGCCAAAAGGCTT
189 CTACGCAGAAGGGAGCAGAGGCGGCAGTCAAGCCTCTTCTCGTTCCTCATCACGTAGTCGCAACAGTTCAAGA
190 AATTCAACTCCAGGCAGCAGTAGGGGAACTTCTCCTGCTAGAATGGCTGGCAATGGCGGTGATGCTGCTCTTG
191 CTTTGCTGCTGCTTGACAGATTGAACCAGCTTGAGAGCAAAATGTCTGGTAAAGGCCAACAACAACAAGGCC
192 AAACTGTCACTAAGAAATCTGCTGCTGAGGCTTCTAAGAAGCCTCGGCAAAAACGTACTGCCACTAAAGCATA
193 CAATGTAACACAAGCTTTCGGCAGACGTGGTCCAGAACAAACCCAAGGAAATTTTGGGGACCAGGAACTAATC
194 AGACAAGGAACTGATTACAAACATTGGCCGCAAATTGCACAATTTGCCCCCAGCGCTTCAGCGTTCTTCGGAAT
195 GTCGCGCATTGGCATGGAAGTCACACCTTCGGGAACGTGGTTGACCTACACAGGTGCCATCAAATTGGATGAC
196 AAAGATCCAAATTTCAAAGATCAAGTCATTTTGCTGAATAAGCATATTGACGCATACAAAACATTCCCACCAAC
197 AGAGCCTAAAAAGGACAAAAAGAAGAAGGCTGATGAAACTCAAGCCTTACCGCAGAGACAGAAGAAACAGC
198 AAACTGTGACTCTTCTTCCTGCTGCAGATTTGGATGATTTCTCCAAACAATTGCAACAATCCATGAGCAGTGCT
199 GACTCAACTCAGGCCTAA
200