Evaluation of PCR-Based Methods for Discrimination of Francisella Species and Subspecies and Development of a Specific PCR That Distinguishes the Two Major Subspecies of Francisella tularensis

Abstract

Previous studies have demonstrated that the four subspecies of the human pathogen Francisella tularensis, despite showing marked variations in their virulence for mammals and originating from different regions in the Northern Hemisphere, display a very close phylogenetic relationship. This property has hampered the development of generally applicable typing methods. To overcome this problem, we evaluated the use of PCR for discrimination of the subspecies using various forms of long arbitrary primers or primers specific for repetitive extragenic palindromic sequences (REP) or enterobacterial repetitive intragenic consensus (ERIC) sequences. Patterns generated by use of REP, ERIC, or long arbitrary primers allowed differentiation at the species level and of the four subspecies of F. tularensis. With each of these three methods, similar or identical clustering of strains was found, and groups of strains of different geographical origins or differing in virulence showed distinct patterns. The discriminatory indices of the methods varied from 0.57 to 0.65; thus, the patterns were not sufficiently discriminatory to distinguish individual strains. The sequence of a fragment generated by amplification with an arbitrary primer was determined, and a region showing interstrain heterogeneity was identified. Specific primers were designed, and a PCR was developed that distinguished strains of F. tularensis subsp.holarctica from strains of other F. tularensissubspecies, including strains of the highly virulent F. tularensis subsp. tularensis. Notably, one European isolate showed the genetic pattern typical of the highly virulentF. tularensis subsp. tularensis, generally believed to exist only in North America. It is proposed that a combination of the specific PCR together with one method generating subspecies-specific patterns is suitable as a rapid and relatively simple strategy for discrimination of Francisella species and subspecies.

The development of tools for diagnosis of tularemia and typing of Francisella tularensis

“…Rapid and clinically relevant characterization is achieved by novel PCR assays that rely on a distinct amplicon size for each subspecies. The first such assay described detects a 30‐bp sequence heterogeneity among the genomes of various F. tularensis strains (26). The PCR assay distinguishes the two clinically most important subspecies, tularensis and holarctica .…”

“…isolates of worldwide origin were grouped into five genotypes representing each of the four subspecies as well as a separate group comprising F. tularensis isolates from Japan. The methods used were repetitive extragenic palindromic sequence (REP)‐PCR, enterobacterial repetitive intragenic consensus sequences (ERIC)‐PCR, and different variations of arbitrary primed (AP)‐PCR (26, 31, 32).…”

Comparison of Closely Related Orthopoxvirus Isolates by Random Amplified Polymorphic DNA and Restriction Fragment Length Polymorphism Analysis

“…These results indicate that even crossing the host barrier from cat to dog or beaver to bearcat obviously did not affect the virus, which showed identical RFLP and RAPD patterns. Variation in the intensities of some of the bands were observed with different PCR runs as already described by Johansson et al (2000) for RAPD analysis of bacteria. For instance the 0.45 kbp band of the strain Bearcat is only weak in the strain Beaver.…”

The utility and perspectives of NGS-based methods in BSL-3 and BSL-4 laboratory – sequencing and analysis strategies

“…Such culturefree diagnostic is very valuable especially in the context of highly pathogenic bacteria like e.g. Bacillus anthracis, Francisella tularensis, Yersinia pestis [1][2][3][4][5] or viruses like e.g. Ebola virus, MERS, SARS, Lassa virus etc.…”