The consensus sequence of E. canis gltA from selected samples was used to generate LAMP primers using the online LAMP primer designing software, Primer Explorer version 5 (http://primerexplorer.jp/e/ ) (Table 1 ).
LAMP reaction mixtures were prepared by mixing 2.5 µL (1×) 10× isothermal amplification buffer, 6 mM MgSO4, 1.4 mM dNTP mix, a primer mix containing 0.2 µM F3/B3 primers, 1.6 µM FIP/BIP primers, and 0.4 µM LF/LB primers, 8 U Bst 2.0 WarmStart® DNA Polymerase (New England Biolabs, Inc., Ipswich, MA, USA), 1 µL colori-fluorometric indicator (CFI), 2 µL DNA, and nuclease-free water to achieve a final volume of 25 µL. CFI contains 3 mM hydroxylnaphthol blue (HNB; MP Biomedicals, Aurora, OH, USA) and 0.35% v/v GelGreen (10 000 × Sol, Biotium, Hayward, CA, USA) dissolved in distilled water [17 (link)]. For optimizing LAMP condition, six PCR-positive and two PCR-negative samples were subjected to a LAMP assay for 60 min with varying temperatures between 60 °C to 65 °C. A negative control (nuclease-free water) was included in each run. The reaction was then terminated by heating at 80 °C for two minutes. After determining the optimum temperature, reaction time was varied to 30, 45, and 60 min.
LAMP reaction mixtures were prepared by mixing 2.5 µL (1×) 10× isothermal amplification buffer, 6 mM MgSO4, 1.4 mM dNTP mix, a primer mix containing 0.2 µM F3/B3 primers, 1.6 µM FIP/BIP primers, and 0.4 µM LF/LB primers, 8 U Bst 2.0 WarmStart® DNA Polymerase (New England Biolabs, Inc., Ipswich, MA, USA), 1 µL colori-fluorometric indicator (CFI), 2 µL DNA, and nuclease-free water to achieve a final volume of 25 µL. CFI contains 3 mM hydroxylnaphthol blue (HNB; MP Biomedicals, Aurora, OH, USA) and 0.35% v/v GelGreen (10 000 × Sol, Biotium, Hayward, CA, USA) dissolved in distilled water [17 (link)]. For optimizing LAMP condition, six PCR-positive and two PCR-negative samples were subjected to a LAMP assay for 60 min with varying temperatures between 60 °C to 65 °C. A negative control (nuclease-free water) was included in each run. The reaction was then terminated by heating at 80 °C for two minutes. After determining the optimum temperature, reaction time was varied to 30, 45, and 60 min.
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