Fig. 2

Colorimetric and real-time LAMP detection of Mannheimia haemolytica genotypes with genotype-specific primers. Colorimetric LAMP reactions were performed using adhesin pseudogene B1 (a, genotype 1) and adhesin G (b, genotype 2) specific primer sets using a non-pH-based multi-purpose LAMP kit supplemented with hydroxynaphthol blue with the purified genomic DNA (5 ng) from M. haemolytica genotypes 1 and 2 strains and four related Pasteurellaceae species at 65 °C for 60 min. A positive reaction is indicated by a color change from violet to sky blue. Similarly, a real-time LAMP amplification of genotype 1 strain (ST2: D171) and genotype 2 strains (ST1: D153 and ST6: D174) was performed with LAMP fluorescent dye at 65 °C for 60 min. Fluorescence readings (SYBR Green I) were recorded every 30 s (c). LAMP amplicons obtained with adhesin pseudogene B1 (a) and adhesin G (b) primers were further analyzed by agarose gel electrophoresis, (d) and (e), respectively. Tubes/lanes information: 1. M. haemolytica genotype 2 (ST1, D153); 2. genotype 1 (ST2, D171); 3. genotype 2 (ST6, D174); 4. Bibersteinia trehalosi; 5. Mannheimia glucosida, 6. Pasteurella multocida; 7. Histophilus somni; 8. No template control. In each gel, molecular weight marker (Bioline HyperLadder 1 kb) is shown to the left of lane 1. GT1: genotype 1, GT2: genotype 2