Francisella tularensis is a Gram-negative bacterium causing Tularemia. While the high pathogenic subspecies Francisella tularensis tularensis has been isolated nearly exclusively in Northern-America the moderate pathogenic subspecies Francisella tularensis holarctica is distributed over the complete northern hemisphere. Especially, Francisella tularensis tularensis has a high potential to be misused as biowarfare agent. Therefore, the assessment of the subspecies of Francisella tularensis is of great importance for patient´s prognosis or as an indication of intentional exposition if Francisella tularensis tularensis is found outside of Northern-America.
To discriminate Francisella tularensis holarctica from other Francisella species and subspecies a SNP polymorphism in the 23s rRNA gene can be used. Besides probe based qPCR systems SNP can be detected by T-Blocker PCR. This technology uses forward primers that end at the SNP position. This is followed by a blocking-primer that binds, if the SNP is not complemetary to the forward primer and prevents amplification. So the amplification is less effective and by comparing ct values of T-Blocker qPCR for both alleles the allele can be distinguished. The set up and validation of this T-Blocker qPCR assay to confirm the occurence of Francisella tularensis holarctica in a sample is presented.