Development of nineteen Taqman real-time PCR assays for screening and detection of select highly pathogenic bacteria

Abstract

Background: Here, we describe a set of six Taqman real-time PCR assays for screening of highly pathogenic bacteria, i.e. Bacillus, Brucella, Burkholderia, Coxiella, Francisella, and Yersinia species. Twelve specific assays are subsequently performed to identify the species that are classified as highly pathogenic and a general 16S Taqman real-time PCR assay is included to see if the sample contains bacteria. Methods: These assays were designed using all available genomes in the public database of bioterror agents. They were validated with a collection of reference strains, clinical isolates and one environmental sample. Results: These assays were tested against all the ring trials we participate among them the ones which were coordinated by Robert Koch Institute from a repository built up in the framework of the EU funded project ‘Efficient response to highly dangerous and emerging pathogens’ (EMERGE). All bacteria were accurately identified in food, clinical and environmental matrices. Conclusions: These assays are used routinely in our diagnostic laboratory to rapidly screen for and specifically detect select highly pathogenic bacteria of potential bioterrorism use. The platform can be used as an open array format in 96-well plates to screen for a single species or up to 6 agents in one run. Abbreviations: ATCC; American Type Culture Collection, B; Brucella; BLAST: Basic local alignment search tool; BSL; Biosafety level; Cq: Quantification cycle; DNA; Deoxyribonucleic acid, FAM; 6-carboxyfluorescein, FOHM; the Public Health Agency of Sweden, IAC; internal amplification control, LOD: Limit of detection, MGB; Minor groove binder, NCBI; National Center for Biotechnology Information, NFA; National Food Agency, PCR; polymerase chain reaction; PhHV-1; Phocine Herpesvirus 1, SVA; National Veterinary Institute, Tm: Melting Temperature.