Table 1. . Reaction mixes for the DNA amplification.
Reagent | Provider | Concentration | Volume, μl | Primer sequence, 5′–3′ | Ref. |
---|---|---|---|---|---|
LAMP | |||||
Primer FIP-F2 | Eurogentec, Germany | 20 μM | 2.25 | GCGCGGCATCCGCATCAATA-TGCCCGGTAAACAGATGAGT | ([16] for the six listed LAMP primers) |
Primer BIP-B2 | Eurogentec, Germany | 20 μM | 2.25 | GCGAACGGCGAAGCGTACTG-TCGCACCGTCAAAGGAAC | – |
Primer B3 | Eurogentec, Germany | 10 μM | 0.25 | CGGCAATAGCGTCACCTT | – |
Primer F3 | Eurogentec, Germany | 10 μM | 0.25 | CGGCCCGATTTTCTCTGG | – |
Primer Loop-F | Eurogentec, Germany | 10 μM | 2.5 | GGCCTTCAAATCGGCATCAAT | – |
Primer Loop-B | Eurogentec, Germany | 10 μM | 2.5 | GAAAGGGAAAGCCAGCTTTACG | – |
MgSO4, molecular biology grade | Sigma Aldrich, Germany | 300 mM | 0.5 | – | |
dNTPs | PeqLab, Germany | 10 mM each | 3 | – | |
Isothermal amplification buffer | New England Biolabs, Germany | 10× | 2.5 | – | |
Bst polymerase | New England Biolabs, Germany | 2 U/μl | 2 | – | |
EvaGreen™ | Biotium, USA | 20× | 1 | – | |
S. enteritidis DNA for positive control | Isolated with QIAamp DNA Mini Kit; Qiagen, Germany | 63.9 ng/μl | 2 | – | |
Water, PCR grade | Jena Bioscience, Germany | - | to 25 μl† | – | |
PCR | |||||
LightCycler® 480 SYBR Green I Master Mix | Roche, Germany | 2× | 10 | – | |
Forward primer uidA | Eurogentec, Germany | 10 μM | 1 | GTTACGTCCTGTAGAAAGCCC | [17,18] |
Reverse primer uidA | Eurogentec, Germany | 10 μM | 1 | AAAACTGCCTGGCACAGCAATT | [17,18] |
E. coli DNA for positive control | Isolated with QIAamp DNA Mini Kit; Qiagen, Germany | 0.5 ng/μl | 1 | – | |
Water, molecular biology grade | Jena Bioscience, Germany | - | to 20 μl† | – |
†Volumes were proportionally scaled up to 200 μl to fill the cartridge lane.
LAMP: Loop-mediated isothermal amplification.