Table 3.
Multiplex LAMP-OSD assay primers, OSD probes, and reaction recipe for detection of SARS-CoV-2.
| Primer/OSD probe name | Sequence |
|---|---|
| NB-F3 | 5’-ACCGAAGAGCTACCAGACG-3’ |
| NB-B3 | 5’-TGCAGCATTGTTAGCAGGAT-3’ |
| NB-FIP | 5’-TCTGGCCCAGTTCCTAGGTAGTTCGTGGTGGTGACGGTAA-3’ |
| NB-BIP | 5’-AGACGGCATCATATGGGTTGCACGGGTGCCAATGTGATCT-3’ |
| NB-LB | 5’-ACTGAGGGAGCCTTGAATACA-3’ |
| NB-OSD-F | /56-FAM/CCG AAT GAA AGA TCT CAG TCC AAG ATG GTA TTT CT/3InvdT/ |
| NB-OSD-Q | 5’-TC TTG GAC TGA GAT CTT TCA TTC GG /3IABkFQ/ |
| Lamb-F3 | 5’-TCCAGATGAGGATGAAGAAGA-3’ |
| Lamb-B3 | 5’-AGTCTGAACAACTGGTGTAAG-3’ |
| Lamb-FIP | 5’-AGAGCAGCAGAAGTGGCACAGGTGATTGTGAAGAAGAAGAG-3’ |
| Lamb-BIP | 5’-TCAACCTGAAGAAGAGCAAGAACTGATTGTCCTCACTGCC-3’ |
| Lamb-LF | 5’-CTCATATTGAGTTGATGGCTCA-3’ |
| Lamb-LB | 5’-ACAAACTGTTGGTCAACAAGAC-3’ |
| Lamb-OSD-F | 5’-GTA TGG TAC TGA AGA TGA TTA CCA AGG TAA ACC TTT GGA ATT TGG AC/36-FAM/ |
| Lamb-OSD-Q | /5IABkFQ/GT CCA AAT TCC AAA GGT TTA CCT TGG TAA TCA TCT C/3InvdT/ |
| Multiplex LAMP-OSD recipe | |
| Component | Volume (μL) per 25 μL reaction |
| RNA or virion templatea | 3 |
| 10 mM dNTP mix | 3.5 |
| NB LAMP primersb | 1.5 |
| Lamb LAMP primersc | 1.5 |
| 10X G6D buffer | 2.5 |
| 5M Betaine | 2 |
| NB OSD probesd | 2.5 |
| Lamb OSD probese | 2.5 |
| 2 × 107 Br512 Mut235 dried bacteria / 3 μL | 3 |
| water | Up to 25 μL |
Use ≥500 copies of RNA or virion templates per reaction.
NB LAMP primers (Zhang et al., 2020) comprise a mix of 25 μM each of NB-FIP and NB-BIP, 10 μM of NB-LB loop primer, and 5 μM each of NB-F3 and NB-B3 primers in TE 10:0.1 buffer.
Lamb LAMP primers (Lamb, Bartolone, Ward, & Chancellor, 2020) comprise a mix of 20 μM each of Lamb-FIP and Lamb-BIP, 10 μM each of Lamb-LB and Lamb-LF loop primers, and 5 μM each of Lamb-F3 and Lamb-B3 primers in TE 10:0.1 buffer.
NB OSD probes are composed of 1 μM of the fluorescein-labeled strand (NB-OSD-F) annealed with 5 μM of the quencher-labeled strand (NB-OSD-Q).
Lamb OSD probes are composed of 1 μM of the fluorescein-labeled strand (Lamb-OSD-F) annealed with 3 μM of the quencher-labeled strand (Lamb-OSD-Q).
Oligonucleotide primers and probes may be purchased from IDT. /3IABkFQ/ and /5IABkFQ/ are the notations used by IDT to represent Iowa Black®FQ dark quencher at the 3’-end or 5’-end, respectively, of an oligonucleotide. /3InvdT/ is the notation used by IDT to represent a 3’-end inverted dT that prevents extension by DNA polymerases. /36-FAM/ and /56-FAM/ are the notations used by IDT to represent a single isomer derivative of fluorescein attached to the 3’-end or 5’-end, respectively, of an oligonucleotide.