Comparison of different PCR approaches for typing of Francisella tularensis strains

Abstract

In this study, we evaluated three PCR methods for epidemiological typing of Francisella tularensis: repetitive extragenic palindromic element PCR (REP-PCR), enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR), and random amplified polymorphic DNA (RAPD) assay with both M13 and T3-T7 primers. The analysis was performed with 40 strains of F. tularensis isolated from hares, humans, ticks, and a vole. On the basis of the combination of REP, ERIC, and RAPD fingerprints, F. tularensis strains were divided into 17 genetic groups (designated A to Q), and one Francisella novicida strain was classified in group R. The F. novicida strain is of special concern, since previous genetic methods have been unable to clearly distinguish between F. tularensis and F. novicida. The F. tularensis isolates recovered from hares were included in groups A to J, M, and P; those recovered from humans were included in groups A, D, G, J, L, O, and N; those isolated from ticks were included in groups B and Q; and that recovered from a vole was in group K. The diversities calculated for the 40 F. tularensis isolates, according to Simpson's index, were 0.14 for REP-PCR, 0.52 for ERIC-PCR, 0.39 for RAPD assay with the M13 primer (RAPD/M13-PCR), and 0.65 for RAPD/T3-T7-PCR, and the diversity increased up to 0.90 when ERIC-PCR, RAPD/M13-PCR, and RAPD/T3-T7-PCR were combined. Our results suggest that although limited genetic heterogeneity among F. tularensis strains was observed, this small variation is enough to validate the PCR methods used in this study and their combinations, because they can provide safe, useful, and rapid tools for the typing of F. tularensis.

FIG. 1

(A) ERIC-PCR fingerprints of type 1 (lane 1), type 2 (lane 2), type 3 (lane 3), and type 4 (lane 4). Lanes M, molecular size marker X (Roche Diagnostics). (B) REP-PCR fingerprints of type 1 (lane 2), type 2 (lane 1), type 3 (lane 3), and type 4 (lane 4). Lanes M, molecular size marker X (Roche Diagnostics). (C) RAPD fingerprints, with the M13 primer, of type 1 (lane 1), type 2 (lane 2), type 3 (lane 3), type 4 (lane 4), and type 5 (lane 5). Lanes M, molecular size marker X (Roche Diagnostics). (D) RAPD fingerprints, with T3-T7 primers, of type 1 (lane 1), type 2 (lane 2), type 3 (lane 3), type 4 (lane 4), type 5 (lane 5), type 6 (lane 6), and type 7 (lane 7). Lanes M, molecular size marker X (Roche Diagnostics).

FIG. 2

Reproducibility studies. The day-to-day reproducibilities of the PCR assays were examined by comparing patterns provided by the same strain and method on four different days. Lanes M, molecular size marker X (Roche Diagnostics). (A) ERIC-PCR fingerprints of strains 27 (type 1) (lanes 1, 2, 3, and 4) and 3 (type 2) (lanes 5, 6, 7, and 8). (B) REP-PCR fingerprints of strains 33 (type 1) (lanes 1, 2, 3, and 4) and 19 (type 2) (lanes 5, 6, 7, and 8).

FIG. 3

Reproducibility studies. The day-to-day reproducibilities of the RAPD/M13 assay were examined by comparing the patterns provided by the five different fingerprints (strain 6, type 1 [lane 1]; strain 19, type 2 [lane 2]; strain 33, type 3 [lane 3]; strain 30, type 4 [lane 4]; and strain 41, type 5 [lane 5]) on four different days (only three days are shown). Lanes M, molecular size marker X (Roche Diagnostics).

FIG. 4

Reproducibility studies. The day-to-day reproducibilities of the RAPD/T3-T7 assay were examined by comparing the patterns provided by the seven different fingerprints (strain 7, type 1 [lane 1]; strain 10, type 2 [lane 2]; strain 20, type 3 [lane 3]; strain 23, type 4 [lane 4]; strain 21, type 5 [lane 5]; strain 28, type 6 [lane 6]; and strain 41, type 7 [lane 7]) on four different days (panels A to D). Lane M, molecular size marker X (Roche Diagnostics).