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Canonical insertion-deletion markers for rapid DNA typing of Francisella tularensis.

Larsson P, Svensson K, Karlsson L, Guala D, Granberg M, Forsman M, Johanssont A - Emerging Infect. Dis. (2007)

Bottom Line: To develop effective and accurate typing of strains of Francisella tularensis, a potent human pathogen and a putative bioterrorist agent, we combined analysis of insertion-deletion (indel) markers with multiple-locus variable-number tandem repeat analysis (MLVA).MLVA included sequences with much diversity in copy number of tandem repeats.The procedure uses limited amounts of killed bacterial preparations and, because only 1 single analytic method is needed, is time- and cost-effective.

View Article: PubMed Central - PubMed

Affiliation: Swedish Defence Research Agency, Umeå, Sweden.

ABSTRACT
To develop effective and accurate typing of strains of Francisella tularensis, a potent human pathogen and a putative bioterrorist agent, we combined analysis of insertion-deletion (indel) markers with multiple-locus variable-number tandem repeat analysis (MLVA). From 5 representative F. tularensis genome sequences, 38 indel markers with canonical properties, i.e., capable of sorting strains into major genetic groups, were selected. To avoid markers with a propensity for homoplasy, we used only those indels with 2 allelic variants and devoid of substantial sequence repeats. MLVA included sequences with much diversity in copy number of tandem repeats. The combined procedure allowed subspecies division, delineation of clades A.I and A.II of subspecies tularensis, differentiation of Japanese strains from other strains of subspecies holarctica, and high-resolution strain typing. The procedure uses limited amounts of killed bacterial preparations and, because only 1 single analytic method is needed, is time- and cost-effective.

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Related in: MedlinePlus

Locations of 38 insertion-deletion and 25 multilocus variable-number tandem repeatanalysis (MLVA) markers on the physical genome map of Francisellatularensis subsp. tularensis strain SCHU S4. Positions aregiven with reference to the predicted origin of replication set at position 0. Indeland MLVA marker locations are depicted by wedges on the outside and inside of thecircle, respectively. Two asterisks indicate the duplicate occurrence of the MLVA lociFt-M14 at 2 different locations because it is part of a large sized genome duplication(1,25).
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Figure 1: Locations of 38 insertion-deletion and 25 multilocus variable-number tandem repeatanalysis (MLVA) markers on the physical genome map of Francisellatularensis subsp. tularensis strain SCHU S4. Positions aregiven with reference to the predicted origin of replication set at position 0. Indeland MLVA marker locations are depicted by wedges on the outside and inside of thecircle, respectively. Two asterisks indicate the duplicate occurrence of the MLVA lociFt-M14 at 2 different locations because it is part of a large sized genome duplication(1,25).

Mentions: PCR amplification was performed in 96-well microtiter plates. Each reaction mixturecontained 0.15 mmol/L dNTP, 0.6 U DyNAzymeII polymerase (F-501L, Finnzymes, Espoo,Finland), 1 μL PCR buffer for DyNAzyme DNA polymerase (Finnzymes), 2μL of template DNA (20 ng/μL), 0.3 pmol/L forward primer, 0.8 pmol/Lreverse primer, and 0.8 pmol/L labeled M13 primer. Filtered sterile water was added to afinal volume of 25 μL. The PCR reactions were performed in a MyCycler thermalcycler (BioRad, Hercules, CA, USA) with the following program: 95°C for 2 min;15 cycles of 95°C for 30 s, 56°C for 30 s, 72°C for 45 s;20 cycles of 95°C for 30 s, 51°C for 30 s, and 72°C for45s; and then a 7-min final extension step at 72°C. MLVA was performed aspreviously described, except modified to use fluorescence-labeled forward primers (6). The physical distribution of 38 selected indel markers identified in this studyand 25 MLVA markers throughout the genome of strain SCHU S4 (29) is illustrated in Figure 1.


Canonical insertion-deletion markers for rapid DNA typing of Francisella tularensis.

Larsson P, Svensson K, Karlsson L, Guala D, Granberg M, Forsman M, Johanssont A - Emerging Infect. Dis. (2007)

Locations of 38 insertion-deletion and 25 multilocus variable-number tandem repeatanalysis (MLVA) markers on the physical genome map of Francisellatularensis subsp. tularensis strain SCHU S4. Positions aregiven with reference to the predicted origin of replication set at position 0. Indeland MLVA marker locations are depicted by wedges on the outside and inside of thecircle, respectively. Two asterisks indicate the duplicate occurrence of the MLVA lociFt-M14 at 2 different locations because it is part of a large sized genome duplication(1,25).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2874433&req=5

Figure 1: Locations of 38 insertion-deletion and 25 multilocus variable-number tandem repeatanalysis (MLVA) markers on the physical genome map of Francisellatularensis subsp. tularensis strain SCHU S4. Positions aregiven with reference to the predicted origin of replication set at position 0. Indeland MLVA marker locations are depicted by wedges on the outside and inside of thecircle, respectively. Two asterisks indicate the duplicate occurrence of the MLVA lociFt-M14 at 2 different locations because it is part of a large sized genome duplication(1,25).
Mentions: PCR amplification was performed in 96-well microtiter plates. Each reaction mixturecontained 0.15 mmol/L dNTP, 0.6 U DyNAzymeII polymerase (F-501L, Finnzymes, Espoo,Finland), 1 μL PCR buffer for DyNAzyme DNA polymerase (Finnzymes), 2μL of template DNA (20 ng/μL), 0.3 pmol/L forward primer, 0.8 pmol/Lreverse primer, and 0.8 pmol/L labeled M13 primer. Filtered sterile water was added to afinal volume of 25 μL. The PCR reactions were performed in a MyCycler thermalcycler (BioRad, Hercules, CA, USA) with the following program: 95°C for 2 min;15 cycles of 95°C for 30 s, 56°C for 30 s, 72°C for 45 s;20 cycles of 95°C for 30 s, 51°C for 30 s, and 72°C for45s; and then a 7-min final extension step at 72°C. MLVA was performed aspreviously described, except modified to use fluorescence-labeled forward primers (6). The physical distribution of 38 selected indel markers identified in this studyand 25 MLVA markers throughout the genome of strain SCHU S4 (29) is illustrated in Figure 1.

Bottom Line: To develop effective and accurate typing of strains of Francisella tularensis, a potent human pathogen and a putative bioterrorist agent, we combined analysis of insertion-deletion (indel) markers with multiple-locus variable-number tandem repeat analysis (MLVA).MLVA included sequences with much diversity in copy number of tandem repeats.The procedure uses limited amounts of killed bacterial preparations and, because only 1 single analytic method is needed, is time- and cost-effective.

View Article: PubMed Central - PubMed

Affiliation: Swedish Defence Research Agency, Umeå, Sweden.

ABSTRACT
To develop effective and accurate typing of strains of Francisella tularensis, a potent human pathogen and a putative bioterrorist agent, we combined analysis of insertion-deletion (indel) markers with multiple-locus variable-number tandem repeat analysis (MLVA). From 5 representative F. tularensis genome sequences, 38 indel markers with canonical properties, i.e., capable of sorting strains into major genetic groups, were selected. To avoid markers with a propensity for homoplasy, we used only those indels with 2 allelic variants and devoid of substantial sequence repeats. MLVA included sequences with much diversity in copy number of tandem repeats. The combined procedure allowed subspecies division, delineation of clades A.I and A.II of subspecies tularensis, differentiation of Japanese strains from other strains of subspecies holarctica, and high-resolution strain typing. The procedure uses limited amounts of killed bacterial preparations and, because only 1 single analytic method is needed, is time- and cost-effective.

Show MeSH
Related in: MedlinePlus