In this study, we evaluated 38 published markers (Table 2) against the current known diversity of the Francisella genus. It is important to note that the studies from which the markers were gathered differed widely in scope. Some studies were designed to only cover a specific species and exclude others, whereas in other studies it was not of interest or even possible to study all the Francisella species included here. Several

of the included markers were amplifying sequence products for species not included in previous studies of Francisella, Tariquidar datasheet e.g. F. hispaniensis, F. noatunensis and W. persica. As many as one third of the markers amplified all the included subspecies and approximately half of the markers

amplified products for F. hispaniensis and/or W. persica together with clade 1 or clade 2. This indicates that strains belonging to F. hispaniensis, W. persica, F. noatunensis are responsible for several false identifications. It should be pointed out that we have only considered sequence based markers here. Other type of markers and marker combinations can be fruitful, in particular for construction of sub-species specific assays, which has been shown by e.g. combining variable-number of tandem repeats (VNTR) and insertion-deletion (indel) markers [35] or SNP and indel markers [36]. Specificity is especially important for markers designed see more for detection. The results of the investigated detection markers suggested that the specificity was questionable for the majority of them. The marker 22-lpnA [37, 38], designated for F. tularensis detection, was found to also amplify F. hispaniensis FSC454 [39]. In the present study, the primers

of the genus-specific marker 13-fopA [16] were not predicted to amplify any of the Molecular motor included F. philomiragia, whereas in the original publication they were reported to amplify all included F. philomiragia isolates. Probably a large unknown diversity exists within this species. For almost all 11 detection markers for Francisella tularensis, there was a significant risk of false-negative results caused by unwanted mismatches for isolates that should be detected. In conclusion, primer sequences need to be continually evaluated and redesigned using up-to date knowledge of the genetic diversity of the targeted sequences to minimise the likelihood of false-positive or -negative results. A similar conclusion was published by [40] where false-positive and -negative hits of primers against publically available sequences in various species of bacteria were evaluated with the result of high degree of primer mismatch in Haemophilus influenza, Pseudomonas aeruginosa and Escherichia coli. Hence, primer miss-match seems to be a general problem within prokaryotes. Our evaluation approach for primers could subsequently be of benefit to the microbiological community.