Molecular grade h2o

The DRB LAMP reaction mix contained the same concentrations of reagents and primers as described for the conventional LAMP assay. However, as the wildtype Bst polymerase large fragment contained glycerol it was not possible to lyophilize. Therefore, a customized Bst polymerase dissolved in H₂O and reaction buffer (NEB) were applied for the DRB LAMP assay. The DRB LAMP reaction mix was prepared for each reaction in 1.5 ml reaction tubes and subjected to lyophilization by means of a RVC 2–25 CD plus Vacuum Concentrator (Christ, Osterode, Germany) at 1.0 mbar and a safety pressure of 1.000 mbar according to the manufacturer’s specifications. During the process of validation, DRB LAMP reaction tubes were stored at ambient temperature in the dark and reactions were carried out within one week after lyophilization as described for cLAMP following the addition of 1 μl template DNA and adjustment with 24 μl molecular grade H₂O (Carl Roth) to a final reaction volume of 25 μl.

Each cLAMP reaction mix consisted of 1 μl Bst DNA polymerase (large fragment, 8 U/μl; New England Biolabs [NEB], Frankfurt am Main, Germany), 1.0 μl dNTP mix (2 mM each, Merck), 1.0 μl of primers MU2-F3 and MU2-B3 (5 pmol/μl) and 2.0 μl of primers MU2-FIP and MU2-BIP (10 pmol/μl), respectively (TibMolBiol, Berlin, Germany), 2.0 μl betaine (5 M; Sigma-Aldrich, Taufkirchen, Germany), 2.5 μl 10-fold Thermopol buffer for Bst DNA polymerase (NEB) and 11.5 μl molecular grade H₂O (Carl Roth). Following the addition of 1 μl DNA extract (template), cLAMP reactions (final volume: 25 μl) were carried out in 1.5 ml SafeSeal reaction tubes (Sarstedt, Nümbrecht, Germany) at 65°C for 60 minutes in a conventional thermoblock (HLC Thermomixer MKR 13, HLC BioTech, Bovenden, Germany) and a final step at 80°C for 10 minutes terminated the amplification.
Each run included negative extraction and no template (H₂O) as well as positive controls.