Real-time PCR for Francisella tularensis Types A and B
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《传染病的形成》
Centers for Disease Control and Prevention, Fort Collins, Colorado, USA
We developed real-time TaqMan PCR assays for classification of F. tularensis type A and type B after F. tularensis is identified by culture or, in the absence of culture, by a PCR method such as the F. tularensis multitarget TaqMan assay (5). The type A TaqMan assay targets pdpD, which is present in type A, almost entirely absent from type B, and contains a 144-bp insert in novicida (6,7) (F: 5′-GAGACATCAATTAAAAGAAGCAATACCTT-3′; R: 5′-CCAAGAGTACTATTTCCGGTTGGT-3′; probe: 5′-AAAATTCTGC"T"CAGCAGGATTTTGATTTGGTT-3′). The type B assay targets a junction between ISFtu2 and a flanking 3′ region (GenBank AY06) (F: 5′- CTTGTACTTTTATTTGGCTACTGAGAAACT-3′; R: 5′- CTTGCTTGGTTTGTAAATATAGTGGAA-3′; probe: 5′- ACCTAGTTCAACC"T"CAAGACTTTTAGTAATGGGAATGTCA-3′). In type A and novicida, ISFtu2 is absent from this position (8). Oligonucleotides were designed with Primer Express version 2.0 (Applied Biosystems, Foster City, CA, USA). Probes were synthesized with a 5′ 6-carboxy-fluorescein reporter and an internal quencher (either BHQ1 [type A] or QSY-7[type B]) at the nucleotide position indicated by the quotation marks.
Assays were optimized by using 1 ng of type A (strain SchuS4) or type B (strain LVS) DNA on the LightCycler 1.2 (Roche Applied Science, Indianapolis, IN, USA). Optimized concentrations (20 μL final volume) were 1× LightCycler Fast Start DNA Master Hybridization Probe mix (Roche), 750 nmol/L primers, 200 nmol/L probe, 5 mmol/L MgCl2 and 0.5 U uracil-DNA glycosylase. PCR conditions were 50°C for 2 min, 95°C for 10 min, 45 cycles of 95°C for 10 s and 65°C for 30 s, then 45°C for 5 min. Cycle threshold (Ct) values were calculated by using the second derivative maximum method with the y-axis at F1/F3 (LightCycler software version 3.5).
Sensitivity of each assay was assessed by using 10-fold serial dilutions (100,000 to 1 genomic equivalents [GE]) of SchuS4 or LVS DNA. Testing was performed in triplicate, with a reproducible detection limit of 10 GE for both assays. Specificity of each assay was tested with 1 ng of DNA from a panel of 62 Francisella isolates (Appendix Table) and 22 non-Francisella isolates (Acinetobacter, Bacillus, Brucella, Corynebacterium, Enterobacter, Enterococcus, Escherichia, Haemophilus, Klebsiella, Legionella, Proteus, Pseudomonas, Serratia, Staphylococcus, Streptococcus, and Yersinia species). Isolates were grown, DNA purified, and quantified as previously described (5). Specificity was also evaluated with DNA (2 μL) extracted as previously described from Francisella-like tick endosymbionts of Dermacentor variabilis and Francisella-like soil bacteria (Appendix Table) (9,10). The type A assay recognized all type A isolates with an average Ct value of 17.9 (n = 19). The type B assay detected all type B strains with an average Ct value of 17.1 (n = 21). Neither assay displayed cross-reactivity with F. tularensis subsp. novicida (n = 7), F. philomiragia (n = 15), Francisella-like tick endosymbionts (n = 3), Francisella-like soil bacteria (n = 7) (Appendix Table), or non-Francisella spp. (n = 22).
To evaluate the ability of the type A and type B TaqMan assays, in conjunction with the multitarget assay, to identify F. tularensis and classify subspecies in primary specimens, human, animal, and tick samples were tested (Table). DNA was extracted from 200 μL fluid, 25 mg liver, and 10 mg spleen or lung by using the QIAamp DNA MiniKit (Qiagen, Valencia, CA, USA) and 1 μL tested. Multitarget PCR conditions were as described (5).
The multitarget and subspecies-specific PCR assays accurately identified and classified F. tularensis in all specimens positive by standard diagnostic methods (Table). In addition, the type A and type B assays provided subspecies information for positive specimens in which an isolate was not recovered for glycerol fermentation testing (Table). All specimens negative by standard diagnostic methods tested negative by PCR. These preliminary results suggest that a F. tularensis PCR identification method, in combination with the type A and type B assays, provides the capability to identify F. tularensis and determine subspecies in the absence of culture.
We describe real-time PCR assays capable of classifying F. tularensis type A and type B and distinguishing these subspecies from the less virulent subsp. novicida. These assays are designed for use after F. tularensis has been identified by culture or by PCR. Supplemental use of these assays will allow laboratories to actively subtype F. tularensis isolates and primary specimens, thus providing subspecies information for a higher percentage of tularemia cases. Improved subspecies information will further understanding of the disease incidence and geographic distribution of F. tularensis type A and type B in North America.
Acknowledgments
We thank Francis Nano for sharing information regarding the pdpD gene; Cheryl Kuske and Susan Barns for sharing DNA from Francisella-like bacteria in soil; and Nikos Gurfield, Jean Creek, and Heidi Goethert for providing Francisella-like tick endosymbiont DNA samples.
References
Ellis J, Oyston PC, Green M, Titball RW. Tularemia. Clin Microbiol Rev. 2002;15:631–46.
Dennis DT, Inglesby TV, Henderson DA, Bartlett JG, Ascher MS, Eitzen E, et al. Tularemia as a biological weapon: medical and public health management. JAMA. 2001;285:2763–73.
Sjostedt A. Family XVII. Francisellaceae, genus I. Francisella. In: Brenner DJ, editor. Bergey’s manual of systematic bacteriology. New York: Springer-Verlag; 2005.
Olsufjev NG, Meshcheryakova IS. Infraspecific taxonomy of tularemia agent Francisella tularensis McCoy et Chapin. J Hyg Epidemiol Microbiol Immunol. 1982;26:291–9.
Versage JL, Severin DD, Chu MC, Petersen JM. Development of a multitarget real-time TaqMan PCR assay for enhanced detection of Francisella tularensis in complex specimens. J Clin Microbiol. 2003;41:5492–9.
Nano FE, Zhang N, Cowley SC, Klose KE, Cheung KK, Roberts MJ, et al. A Francisella tularensis pathogenicity island required for intramacrophage growth. J Bacteriol. 2004;186:6430–6.
Larsson P, Oyston PC, Chain P, Chu MC, Duffield M, Fuxelius HH, et al. The complete genome sequence of Francisella tularensis, the causative agent of tularemia. Nat Genet. 2005;37:153–9.
Petersen JM, Schriefer ME, Carter LG, Zhou Y, Sealy T, Bawiec D, et al. Laboratory analysis of tularemia in wild-trapped, commercially traded prairie dogs, Texas, 2002. Emerg Infect Dis. 2004;10:419–25.
Barns SM, Grow CC, Okinaka RT, Keim P, Kuske CR. Detection of diverse new Francisella-like bacteria in environmental samples. Appl Environ Microbiol. 2005;71:5494–500.
Kugeler KJ, Gurfield N, Creek JG, Mahoney KS, Versage JL, Petersen JM. Discrimination between Francisella tularensis and Francisella-like endosymbionts when screening ticks by PCR. Appl Environ Microbiol. 2005;71:7594–7.(Kiersten J. Kugeler, Ryan)
We developed real-time TaqMan PCR assays for classification of F. tularensis type A and type B after F. tularensis is identified by culture or, in the absence of culture, by a PCR method such as the F. tularensis multitarget TaqMan assay (5). The type A TaqMan assay targets pdpD, which is present in type A, almost entirely absent from type B, and contains a 144-bp insert in novicida (6,7) (F: 5′-GAGACATCAATTAAAAGAAGCAATACCTT-3′; R: 5′-CCAAGAGTACTATTTCCGGTTGGT-3′; probe: 5′-AAAATTCTGC"T"CAGCAGGATTTTGATTTGGTT-3′). The type B assay targets a junction between ISFtu2 and a flanking 3′ region (GenBank AY06) (F: 5′- CTTGTACTTTTATTTGGCTACTGAGAAACT-3′; R: 5′- CTTGCTTGGTTTGTAAATATAGTGGAA-3′; probe: 5′- ACCTAGTTCAACC"T"CAAGACTTTTAGTAATGGGAATGTCA-3′). In type A and novicida, ISFtu2 is absent from this position (8). Oligonucleotides were designed with Primer Express version 2.0 (Applied Biosystems, Foster City, CA, USA). Probes were synthesized with a 5′ 6-carboxy-fluorescein reporter and an internal quencher (either BHQ1 [type A] or QSY-7[type B]) at the nucleotide position indicated by the quotation marks.
Assays were optimized by using 1 ng of type A (strain SchuS4) or type B (strain LVS) DNA on the LightCycler 1.2 (Roche Applied Science, Indianapolis, IN, USA). Optimized concentrations (20 μL final volume) were 1× LightCycler Fast Start DNA Master Hybridization Probe mix (Roche), 750 nmol/L primers, 200 nmol/L probe, 5 mmol/L MgCl2 and 0.5 U uracil-DNA glycosylase. PCR conditions were 50°C for 2 min, 95°C for 10 min, 45 cycles of 95°C for 10 s and 65°C for 30 s, then 45°C for 5 min. Cycle threshold (Ct) values were calculated by using the second derivative maximum method with the y-axis at F1/F3 (LightCycler software version 3.5).
Sensitivity of each assay was assessed by using 10-fold serial dilutions (100,000 to 1 genomic equivalents [GE]) of SchuS4 or LVS DNA. Testing was performed in triplicate, with a reproducible detection limit of 10 GE for both assays. Specificity of each assay was tested with 1 ng of DNA from a panel of 62 Francisella isolates (Appendix Table) and 22 non-Francisella isolates (Acinetobacter, Bacillus, Brucella, Corynebacterium, Enterobacter, Enterococcus, Escherichia, Haemophilus, Klebsiella, Legionella, Proteus, Pseudomonas, Serratia, Staphylococcus, Streptococcus, and Yersinia species). Isolates were grown, DNA purified, and quantified as previously described (5). Specificity was also evaluated with DNA (2 μL) extracted as previously described from Francisella-like tick endosymbionts of Dermacentor variabilis and Francisella-like soil bacteria (Appendix Table) (9,10). The type A assay recognized all type A isolates with an average Ct value of 17.9 (n = 19). The type B assay detected all type B strains with an average Ct value of 17.1 (n = 21). Neither assay displayed cross-reactivity with F. tularensis subsp. novicida (n = 7), F. philomiragia (n = 15), Francisella-like tick endosymbionts (n = 3), Francisella-like soil bacteria (n = 7) (Appendix Table), or non-Francisella spp. (n = 22).
To evaluate the ability of the type A and type B TaqMan assays, in conjunction with the multitarget assay, to identify F. tularensis and classify subspecies in primary specimens, human, animal, and tick samples were tested (Table). DNA was extracted from 200 μL fluid, 25 mg liver, and 10 mg spleen or lung by using the QIAamp DNA MiniKit (Qiagen, Valencia, CA, USA) and 1 μL tested. Multitarget PCR conditions were as described (5).
The multitarget and subspecies-specific PCR assays accurately identified and classified F. tularensis in all specimens positive by standard diagnostic methods (Table). In addition, the type A and type B assays provided subspecies information for positive specimens in which an isolate was not recovered for glycerol fermentation testing (Table). All specimens negative by standard diagnostic methods tested negative by PCR. These preliminary results suggest that a F. tularensis PCR identification method, in combination with the type A and type B assays, provides the capability to identify F. tularensis and determine subspecies in the absence of culture.
We describe real-time PCR assays capable of classifying F. tularensis type A and type B and distinguishing these subspecies from the less virulent subsp. novicida. These assays are designed for use after F. tularensis has been identified by culture or by PCR. Supplemental use of these assays will allow laboratories to actively subtype F. tularensis isolates and primary specimens, thus providing subspecies information for a higher percentage of tularemia cases. Improved subspecies information will further understanding of the disease incidence and geographic distribution of F. tularensis type A and type B in North America.
Acknowledgments
We thank Francis Nano for sharing information regarding the pdpD gene; Cheryl Kuske and Susan Barns for sharing DNA from Francisella-like bacteria in soil; and Nikos Gurfield, Jean Creek, and Heidi Goethert for providing Francisella-like tick endosymbiont DNA samples.
References
Ellis J, Oyston PC, Green M, Titball RW. Tularemia. Clin Microbiol Rev. 2002;15:631–46.
Dennis DT, Inglesby TV, Henderson DA, Bartlett JG, Ascher MS, Eitzen E, et al. Tularemia as a biological weapon: medical and public health management. JAMA. 2001;285:2763–73.
Sjostedt A. Family XVII. Francisellaceae, genus I. Francisella. In: Brenner DJ, editor. Bergey’s manual of systematic bacteriology. New York: Springer-Verlag; 2005.
Olsufjev NG, Meshcheryakova IS. Infraspecific taxonomy of tularemia agent Francisella tularensis McCoy et Chapin. J Hyg Epidemiol Microbiol Immunol. 1982;26:291–9.
Versage JL, Severin DD, Chu MC, Petersen JM. Development of a multitarget real-time TaqMan PCR assay for enhanced detection of Francisella tularensis in complex specimens. J Clin Microbiol. 2003;41:5492–9.
Nano FE, Zhang N, Cowley SC, Klose KE, Cheung KK, Roberts MJ, et al. A Francisella tularensis pathogenicity island required for intramacrophage growth. J Bacteriol. 2004;186:6430–6.
Larsson P, Oyston PC, Chain P, Chu MC, Duffield M, Fuxelius HH, et al. The complete genome sequence of Francisella tularensis, the causative agent of tularemia. Nat Genet. 2005;37:153–9.
Petersen JM, Schriefer ME, Carter LG, Zhou Y, Sealy T, Bawiec D, et al. Laboratory analysis of tularemia in wild-trapped, commercially traded prairie dogs, Texas, 2002. Emerg Infect Dis. 2004;10:419–25.
Barns SM, Grow CC, Okinaka RT, Keim P, Kuske CR. Detection of diverse new Francisella-like bacteria in environmental samples. Appl Environ Microbiol. 2005;71:5494–500.
Kugeler KJ, Gurfield N, Creek JG, Mahoney KS, Versage JL, Petersen JM. Discrimination between Francisella tularensis and Francisella-like endosymbionts when screening ticks by PCR. Appl Environ Microbiol. 2005;71:7594–7.(Kiersten J. Kugeler, Ryan)