PCR amplification of fur with various PCR mix and cycle parameters (DNA concentration, MgCl concentration, elongation time, number of cycles, annealing temperature) were tested for our strains in our laboratory and by an independent service provider (Labofarm). Tests were also conducted using the ready-made PCR mix recommended by the authors of the fur identification method (TEMPase Hot Start Master Mix Blue (Ampliqon A/S, Odense, Denmark)). To test if fur sequence was amplified after PCR assay, a PCR amplification targeted at a fragment of fur was conducted as recommended by Machado and Gram, 2015 (link), which turned out negative except for 2 of the 25 strains used for testing. During this testing steps, negative controls where DNA was replaced by ultrapure water were executed. Positive controls using the strains that allowed PCR amplification of fur were also executed.
Our assay and the assays conducted by Labofarm concluded independently that the primers described in the literature were too degenerate, as most of the strains were resistant to fur PCR amplification. It was therefore decided to use WGS data to extract fur. Fur based identification was performed using FurIOS webserver (Machado et al., 2017) (link). For ldh identification, the gene was also retrieved after genome annotation.
Our assay and the assays conducted by Labofarm concluded independently that the primers described in the literature were too degenerate, as most of the strains were resistant to fur PCR amplification. It was therefore decided to use WGS data to extract fur. Fur based identification was performed using FurIOS webserver (Machado et al., 2017) (link). For ldh identification, the gene was also retrieved after genome annotation.