Real-time PCR for Francisella tularensis types A and B.To the Editor: Francisella tularensis Francisella tu·la·ren·sis n. A bacterium of the genus Francisella that causes tularemia in humans. , the etiologic agent of tularemia tularemia (t  lərē`mēə) or rabbit fever, acute, infectious disease caused by Francisella tularensis (Pasteurella tularensis). , is highly infectious and considered a potential bioweapon Noun 1. bioweapon - any weapon usable in biological warfare; "they feared use of the smallpox virus as a bioweapon"bioarm, biological weapon anthrax bacillus, Bacillus anthracis - a species of bacillus that causes anthrax in humans and in animals (cattle (1-3). Although 4 subspecies subspecies, also called race, a genetically distinct geographical subunit of a species. See also classification. of F. tularensis are recognized, most cases of tularemia are due to infection by subsp, tularensis (type A) or holarctica (type B). North America is the only region where both type A and type B cause human disease. Subspecies novicida is also found in North America, but it is of reduced virulence. Disease incidence attributable to either type A or type B is essentially unknown because the traditional method for classification of these subspecies is glycerol glycerol, glycerin, glycerine, or 1,2,3-propanetriol (prō`pāntrī'ŏl), CH2OHCHOHCH2OH, colorless, odorless, sweet-tasting, syrupy liquid. fermentation, which requires culture recovery (4). F. tularensis is fastidious fas·tid·i·ous adj. 1. Possessing or displaying careful, meticulous attention to detail. 2. Difficult to please; exacting. 3. Having complex nutritional requirements. Used of microorganisms. and slow growing, with isolates recovered in a small percentage of cases. We developed real-time TaqMan PCR PCR polymerase chain reaction. PCR abbr. polymerase chain reaction Polymerase chain reaction (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'GAGACATCAATTAAAAGAAG- CAATACCTT-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'CTTGTACTTTTATTTGGCTACT- GAGAAACT-3'; R: 5'- CTTGCTTGGTTTGTAAATATAGTGGAA-3'; probe: 5'- ACCTAGTTCAACC"T"CAAGACTTTTAGTAATGGGAAT- GTCA-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 quench tr.v. quenched, quench·ing, quench·es 1. To put out (a fire, for example); extinguish. 2. To suppress; squelch: (either BHQ BHQ Biblia Hebraica Quinta (Hebrew Bible) BHQ Broken Hill, New South Wales, Australia - Broken Hill (Airport Code) BHQ Brent Hartman Billiard Cues 1 [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 LVS Linux Virtual Server LVS Live Vaccine Strain LVS Las Vegas, New Mexico (Airport Code) LVS Low Voltage Switchgear LVS Logistical Vehicle System LVS Laser Vibration Sensor LVS Logistics Vehicle System ) DNA DNA: see nucleic acid. DNA or deoxyribonucleic acid One of two types of nucleic acid (the other is RNA); a complex organic compound found in all living cells and many viruses. It is the chemical substance of genes. on the LightCycler 1.2 (Roche Applied Science, Indianapolis, IN, USA). Optimized concentrations (20 p.L final volume) were lx LightCycler Fast Start DNA Master Hybridization Probe mix (Roche), 750 nmol/L primers, 200 nmol/L probe, 5 mmol/L Mg[Cl.sub.2] and 0.5 U uracil-DNA glycosylase. PCR conditions were 50[degrees]C for 2 min, 95[degrees]C for 10 min, 45 cycles of 95[degrees]C for 10 s, and 65[degrees]C for 30 s, then 45[degrees]C for 5 min. Cycle threshold ([C.sub.t]) 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 (online Appendix Table, available from http://www.cdc.gov/ ncidod/EID/vol 12no 11/06-0629 appT.htm) and 22 non-Francisella isolates (Acinetobacter, Bacillus bacillus (bəsĭl`əs), any rod-shaped bacterium or, more particularly, a rod-shaped bacterium of the genus Bacillus. Some bacterium in the genus cause disease, for example B. , Brucella Brucella /Bru·cel·la/ (broo-sel´ah) a genus of schizomycetes (family Brucellaceae). B. abor´tus causes infectious abortion in cattle and is the most common cause of brucellosis in humans. B. , Corynebacterium Corynebacterium /Co·ry·ne·bac·te·ri·um/ (-bak-ter´e-um) a genus of bacteria including C. ac´nes, a species present in acne lesions, C. diphthe´riae, the etiologic agent of diphtheria, C. , Enterobacter, Enterococcus enterococcus /en·tero·coc·cus/ (en?ter-o-kok´us) pl. enterococ´ci an organism belonging to the genus Enterococcus. Enterococcus /En·tero·coc·cus/ ( , Escherichia, Haemophilus, Klebsiella klebsiella Any of the rod-shaped bacteria that make up the genus Klebsiella. They are gram-negative (see gram stain), thrive better without oxygen than with it, and do not move. K. , Legionella Legionella /Le·gion·el·la/ (le?jah-nel´ah) a genus of gram-negative, aerobic, rod-shaped bacteria (family Legionellaceae), normal inhabitants of lakes, streams, and moist soil; they have often been isolated from cooling-tower water, , Proteus, Pseudomonas Pseudomonas A genus of gram-negative, nonsporeforming, rod-shaped bacteria. Motile species possess polar flagella. They are strictly aerobic, but some members do respire anaerobically in the presence of nitrate. , Serratia, Staphylococcus staphylococcus (stăf'ələkŏk`əs), any of the pathogenic bacteria, parasitic to humans, that belong to the genus Staphylococcus. The spherical bacterial cells (cocci) typically occur in irregular clusters [Gr. , Streptococcus streptococcus (strĕp'təkŏk`əs), any of a group of gram-positive bacteria, genus Streptococcus, some of which cause disease. , and Yersinia Yersinia A genus of bacteria in the Enterobacteriaceae family. The bacteria appear as gram-negative rods and share many physiological properties with related Escherichia coli. Of the 11 species of Yersinia, Y. pestis, Y. enterocolitica, and Y. species). Isolates were grown, DNA purified, and quantified as previously described (5). Specificity was also evaluated with DNA (2 [micro]L) extracted as previously described from Francisella-like tick endosymbionts of Dermacentor variabilis Dermacentor var·i·a·bi·lis n. A tick that transmits tularemia and is the principal vector of Rocky Mountain spotted fever in the central and eastern US; the American dog tick. and Francisella-like soil bacteria (online Appendix) (9,10). The type A assay recognized all type A isolates with an average [C.sub.t] value of 17.9 (n - 19). The type B assay detected all type B strains with an average [C.sub.t] 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), Francisellalike soil bacteria (n = 7) (Appendix), 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) available from DNA was extracted from 200 [micro]L fluid, 25 mg liver, and 10 mg spleen or lung by using the QIAamp DNA MiniKit (Qiagen, Valencia, CA, USA) and 1 [micro]L 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 (programming) subtype - If S is a subtype of T then an expression of type S may be used anywhere that one of type T can and an implicit type conversion will be applied to convert it to type T. 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 An endosymbiont is any organism that lives within the body or cells of another organism, i.e. forming an endosymbiosis (Greek: endo = inner, sym = together and biosis = living). DNA samples. References (1.) Ellis J, Oyston PC, Green M, Titball RW. Tularemia. Clin Microbiol Rev. 2002;15:631-46. (2.) 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 JAMA abbr. Journal of the American Medical Association . 2001 ;285:2763-73. (3.) Sjostedt A. Family XVII. Francisellaceae, genus I. Francisella. In: Brenner DJ, editor. Bergey's manual of systematic bacteriology bacteriology Study of bacteria. Modern understanding of bacterial forms dates from Ferdinand Cohn's classifications. Other researchers, such as Louis Pasteur, established the connection between bacteria and fermentation and disease. . New York New York, state, United States New York, Middle Atlantic state of the United States. It is bordered by Vermont, Massachusetts, Connecticut, and the Atlantic Ocean (E), New Jersey and Pennsylvania (S), Lakes Erie and Ontario and the Canadian province of : Springer-Verlag; 2005. (4.) Olsufjev NG, Meshcheryakova IS. Infraspecific in·fra·spe·cif·ic adj. Occurring within a species. infraspecific Occurring within a species; intraspecific. taxonomy of tularemia agent Francisella tularensis McCoy et Chapin. J Hyg Epidemiol Microbiol Immunol. 1982;26:2914. (5.) 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. (6.) 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. (7.) 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 genet: see civet. . 2005;37:153-9. (8.) 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. (9.) 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. (10.) 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 Pappert, * Yan Zhou, * and Jeannine M. Petersen * * Centers for Disease Control and Prevention Centers for Disease Control and Prevention (CDC), agency of the U.S. Public Health Service since 1973, with headquarters in Atlanta; it was established in 1946 as the Communicable Disease Center. , Fort Collins, Colorado The City of Fort Collins, a home rule municipality situated on the Cache la Poudre River along the Colorado Front Range, is the county seat and most populous city in Larimer County, Colorado. , USA Address for correspondence: Jeannine M. Petersen, Division of Vector-Borne infectious Diseases, Centers for Disease Control and Prevention, Foothills Campus, PO Box 2087, Fort Collins, CO 80522, USA; email: nzp0@cdc.gov
Table. Comparison of standard diagnostic methods with the multitarget
Francisella tularensis TaqMan assay and type A and type B assays
using primary specimens
Subspecies
F. tularensis identification
Specimen Source identified * ([dagger])
Lymph node aspirate Human +
Bronchial wash Human + A
Upper lung Human + A
Lower lung Human + A
Liver Human + A
Spleen Human + A
Pleural fluid Human + B
Blood Human +
Spleen Human -
Liver Human -
Cerebrospinal fluid Human -
Blood Human -
Liver/spleen Tamarin +
Tissue Tick ([paragraph]) + A
Tissue Tick ([paragraph]) + A
Blood Prairie dog + B
Blood Prairie dog + B
Spleen Prairie dog + B
Spleen Prairie dog + B
Spleen Prairie dog -
Liver Cat -
Liver Rat -
Spleen Rat -
Spleen Squirrel -
Multitarget F. tularensis
TaqMan assay ([double dagger])
Specimen ISFtu2 iglC tul4
Lymph node aspirate + + +
Bronchial wash + + +
Upper lung + + +
Lower lung + + +
Liver + + +
Spleen + + +
Pleural fluid + + +
Blood + + +
Spleen - - -
Liver - - -
Cerebrospinal fluid - - -
Blood - - -
Liver/spleen + + +
Tissue + + +
Tissue + + +
Blood + + +
Blood + + +
Spleen + + +
Spleen + + +
Spleen - - -
Liver - - -
Liver - - -
Spleen - - -
Spleen - - -
Type A assay Type B assay
([double dagger]) ([double dagger])
([C.sub.t] value) ([C.sub.t]
Specimen ([section]) value)
Lymph node aspirate 31 -
Bronchial wash 34 -
Upper lung 20 -
Lower lung 26 -
Liver 29 -
Spleen 31 -
Pleural fluid - 36
Blood - 38
Spleen - -
Liver - -
Cerebrospinal fluid - -
Blood - -
Liver/spleen 28 -
Tissue 26 -
Tissue 33 -
Blood - 30
Blood - 27
Spleen - 21
Spleen - 31
Spleen - -
Liver - -
Liver - -
Spleen - -
Spleen - -
* F. tularensis infection identified by culture, direct fluorescent
antibody testing, or serologic testing.
([dagger]) Subspecies was determined by glycerol fermentation when
an isolate was recovered.
([double dagger]) + = positive result, 17 [less than or equal to]
[C.sub.t] [less than or equal to] 38; - = negative result, no
fluorescence detected after 45 cycles of amplification.
([section]) [C.sub.t], cycle threshold.
([paragraph]) Tick species tested were Haemaphysalis leporispalustris
and Dermacentorandersoni.
|
|
||||||||||||||||||
Printer friendly
Cite/link
Email
Feedback
Reader Opinion