a real-time pcr array for hierarchical identification of francisella isolates实时pcr阵列分层标识隔离.pdfVIP

A Real-Time PCR Array for Hierarchical Identification of Francisella Isolates 1,2 1 1 3 1 Kerstin Svensson *, Malin Granberg , Linda Karlsson , Vera Neubauerova , Mats Forsman , Anders Johansson1,2 ˚ 1 Division of CBRN Defense and Security, Swedish Defense Research Agency, Umea, Sweden, 2 Department of Clinical Microbiology, Infectious Diseases and Bacteriology, ˚ ˚ Umea University, Umea, Sweden, 3 Central Military Health Institute, Prague, Czech Republic Abstract A robust, rapid and flexible real-time PCR assay for hierarchical genetic typing of clinical and environmental isolates of Francisella is presented. Typing markers were found by multiple genome and gene comparisons, from which 23 canonical single nucleotide polymorphisms (canSNPs) and 11 canonical insertion-deletion mutations (canINDELs) were selected to provide phylogenetic guidelines for classification from genus to isolate level. The specificity of the developed assay, which uses 68 wells of a 96-well real-time PCR format with a detection limit of 100 pg DNA, was assessed using 62 Francisella isolates of diverse genetic and geographical origins. It was then successfully used for typing 14 F. tularensis subsp. holarctica isolates obtained from tularemia patients in Sweden in 2008 and five more genetically diverse Francisella isolates of global origins. When applied to human ulcer specimens for direct pathogen detection the results were incomplete due to scarcity of DNA, but sufficient markers were identified to detect fine-resolution differences among F. tularensis subsp. holarctica isolates causing infection in the