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SARS Epidemiology
Research
SARS Surveillance during Emergency
Public Health Response, United States, March–July 2003
Stephanie J. Schrag,* John T. Brooks,* Chris Van Beneden,* Umesh D.
Parashar,* Patricia M. Griffin,* Larry J. Anderson,* William J. Bellini,*
Robert F. Benson,* Dean D. Erdman,* Alexander Klimov,* Thomas G. Ksiazek,*
Teresa C.T. Peret,* Deborah F. Talkington,* W. Lanier Thacker,* Maria
L. Tondella,* Jacquelyn S. Sampson,* Allen W. Hightower,* Dale F. Nordenberg,*
Brian D. Plikaytis,* Ali S. Khan,* Nancy E. Rosenstein,* Tracee A. Treadwell,*
Cynthia G. Whitney,* Anthony E. Fiore,* Tonji M. Durant,* Joseph F. Perz,*
Annemarie Wasley,* Daniel Feikin,* Joy L. Herndon,* William A. Bower,*
Barbara W. Kilbourn,* Deborah A. Levy,* Victor G. Coronado,* Joanna Buffington,*
Clare A. Dykewicz,* Rima F. Khabbaz,* and Mary E. Chamberland*
*Centers for Disease Control and Prevention, Atlanta, Georgia, USA
Appendix
Legionella pneumophila
Real-Time PCR Assay
Primers and a fluorescent probe were based on the nucleotide sequences
of the 5S RNA gene of Legionella pneumophila serogroup 1 for a
genus-specific assay. Primers for the 5S gene target were as follows:
5´-GCG ACT ATA GCG ITI TGG AA-3´ (forward primer) and 5´-CGA
TGA CCT ACT TTC ICA TGA-3´ (reverse primer). The probe sequence
was 5´-Fam-TC ACA CTA TCA TIG GCG CGG AAA QSY7-3´. They were
designed by using ABI Primer Express software (Applied Biosystems, Foster
City, CA). The fluorogenic probes were synthesized with a FAM (6-carboxy-fluorescein)
reporter molecule attached at the 5´ end and a QSY7 NHS ester quencher
dye linked to a linker arm nucleotide phosphoramidite (Glen Research,
Sterling, VA) close to the 3´ end. The probes were synthesized with
a 3´ terminal phosphate group to prevent extension during PCR. Primers
and probes were synthesized in the Biotechnology Core Facility at CDC
by using model 394-8 DNA synthesizer and standard phosphoramidite chemistries
(Applied Biosystems, Foster City, CA).
Reactions were prepared in a 96-well MicroAmp optical plate (Applied
Biosystems) for the 5S RNA assay by addition of a 5-μL aliquot of
extracted DNA to 20 μL of a PCR master mixture consisting of 1X TaqMan
2X universal PCR master mix (Applied Biosystems), 300 nM each primer,
and 100 nM fluorescent-labeled probe. Primers and probes were previously
titrated to check for amplification efficiency. The amplification and
detection were performed with an AB Prism 7700 Sequence Detection System
(Applied Biosystems). The thermal cycle conditions were: 50°C for 2 min,
95°C for 10 min, and 40 cycles of 95°C for 15 s, and 60°C for 1 min. Standard
procedures for the operation of model 7700 were followed in this study,
including the use of all default program settings with the exception of
reaction volume, which was changed from 50 mL to 25 mL. Cycle threshold
(CT) values, defined as the fraction of a cycle number at which
the measured fluorescence generated by the released reporter molecule
during cleavage exceeds a fixed threshold value above baseline, were automatically
calculated by the instrument for each reaction. Target gene copy values
were derived from a standard curve generated by plotting the CT
values of a dilution curve that consisted of 10-fold serial dilutions
ranging from 7.5 × 106 to 7.5 × 100 copies. Each
run contained at least four no-template controls to establish the baseline
emission intensity of the quenched reporter dye. Negative controls (one
for every five DNA extracted samples) were included. Specificity was determined
by testing DNA extracted from other bacterial species that are commonly
found in the human respiratory tract. Amplification plots were compared
with the ones obtained by using only the serial dilutions of the L.
pneumophila isolate as DNA templates.
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