Table 4.
LAMP-OSD assay primers, OSD probes, and reaction recipe for detection of human glyceraldehyde 3-phosphate-dehydrogenase gene.
| Primer/OSD probe name | Sequence |
|---|---|
| hGAPD.F3 | 5’-GCCACCCAGAAGACTGTG-3’ |
| hGAPD.B3 | 5’-TGGCAGGTTTTTCTAGACGG-3’ |
| hGAPD.FIP | 5’-CGCCAGTAGAGGCAGGGATGAGGGAAACTGTGGCGTGAT-3’ |
| hGAPD.BIP | 5’-GGTCATCCCTGAGCTGAACGGTCAGGTCCACCACTGACAC-3’ |
| hGAPD.LR | 5’-TGTTCTGGAGAGCCCCGCGGCC-3’ |
| hGAPD.OSD.F | /56-FAM/CTCACTGGCATGGCCTTCCGTGTCCCCACTGCCAAC/3InvdT/ |
| hGAPD.OSD.Q | 5’-GGACACGGAAGGCCATGCCAGTGAG/3IABkFQ/ |
| GAPD LAMP amplicon sequence | 5’-GCCACCCAGAAGACTGTGGATGGCCCCTCCGGGAAACTGTGGCGTGATGGCCGCGGGGCTCTCCAGAACATCATCCCTGCCTCTACTGGCGCTGCCAAGGCTGTGGGCAAGGTCATCCCTGAGCTGAACGGGAAGCTCACTGGCATGGCCTTCCGTGTCCCCACTGCCAACGTGTCAGTGGTGGACCTGACCTGCCGTCTAGAAAAACCTGCCA-3’ |
| LAMP-OSD recipe | |
| Component | Volume (μL) per 25 μL reaction |
| DNA templatea | 5 |
| 10X G6B buffer | 2.5 |
| 4 mM dNTP mix | 2.5 |
| LAMP primer mixb | 2 |
| OSD probesc | 2.5 |
| 5 M Betaine | 5 |
| 2 × 107 Br512 Mut235 dried bacteria / 3 μL | 3 |
| Water | Up to 25 μL |
Use ≥600 copies of DNA templates per reaction.
LAMP primer mix comprises 20 μM each of hGAPD.FIP and hGAPD.BIP, 10 μM of hGAPD.LR loop primer, and 5 μM each of hGAPD.F3 and hGAPD.B3 primers in TE 10:0.1 buffer.
OSD probes are composed of 1 μM of the fluorescein-labeled strand (hGAPD.OSD.F) annealed with 2 μM of the quencher-labeled strand (hGAPD.OSD.Q).
Oligonucleotide primers and probes and double stranded DNA gBlock™ or eBlock™ templates may be purchased from IDT. /3IABkFQ/ is the notation used by IDT to represent Iowa Black®FQ dark quencher at the 3’-end of an oligonucleotide. /3InvdT/ is the notation used by IDT to represent a 3’-end inverted dT that prevents extension by DNA polymerases. /56-FAM/ is the notation used by IDT to represent a single isomer derivative of fluorescein attached to the 5’-end of an oligonucleotide.