Evaluation of PCR-based methods for discrimination of Francisella species and subspecies and development of a specific PCR that distinguishes the twomajor subspecies of Francisella tularensis
A. Johansson et al., Evaluation of PCR-based methods for discrimination of Francisella species and subspecies and development of a specific PCR that distinguishes the twomajor subspecies of Francisella tularensis, J CLIN MICR, 38(11), 2000, pp. 4180-4185
Previous studies have demonstrated that the four subspecies of the human pa
thogen Francisella tularensis, despite showing marked variations in their v
irulence 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 ove
rcome this problem, we evaluated the use of PCR for discrimination of the s
ubspecies using various forms of long arbitrary primers or primers specific
for repetitive extragenic palindromic sequences (REP) or enterobacterial r
epetitive intragenic consensus (ERIC) sequences. Patterns generated by use
of REP, ERIC, or long arbitrary primers allowed differentiation at the spec
ies level and of the four subspecies of F. tularensis. With each of these t
hree methods, similar or identical clustering of strains was found, and gro
ups 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 off. tularensis subsp. h
olarctica from strains of other F. tularensis subspecies. including strains
of the highly virulent F. tularensis subsp. tularensis, Notably, one Europ
ean isolate showed the genetic pattern typical of the highly virulent F. tu
larensis subsp. tularensis, generally believed to exist only in North Ameri
ca. 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 su
bspecies.