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Rapid Genotyping of Bacillus anthracis Strains by Real‐Time Polymerase Chain Reaction
Author(s) -
PATRA GUY,
WILLIAMS LEANNE E.,
QI YUAN,
ROSE SHARON,
REDKAR RAJENDRA,
DELVECCHIO VITO G.
Publication year - 2002
Publication title -
annals of the new york academy of sciences
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.712
H-Index - 248
eISSN - 1749-6632
pISSN - 0077-8923
DOI - 10.1111/j.1749-6632.2002.tb04360.x
Subject(s) - bacillus anthracis , förster resonance energy transfer , primer (cosmetics) , microbiology and biotechnology , genotyping , fluorescein , polymerase chain reaction , denaturation (fissile materials) , lysis , chemistry , fluorescence , plasmid , biology , real time polymerase chain reaction , primer dimer , dna , genotype , biochemistry , genetics , multiplex polymerase chain reaction , gene , bacteria , physics , organic chemistry , quantum mechanics , nuclear chemistry
A bstract : Rapid and accurate identification of Bacillus anthracis is critical for patient care as well as outbreak control. We have developed 3 separate PCR based assays using fluorescence resonance energy transfer (FRET) to detect the presence of pXO1, pXO2 plasmids and a chromosomal marker. A set of amplification primers and probes were used in each assay. The probes were ad jacently placed inside the primer sites and were 1‐bp apart. The upstream probe was labeled with fluorescein at the 3′ end, and the downstream probe had Cy5 attached at the 5′ end. The probes are included in the PCR reactions and hybridize with the PCR products as they are formed. Binding of probes to PCR products results in transfer of energy from fluorescein to Cy5, resulting in emission from Cy5. Increase in fluorescence, indicating amplification, was monitored in real time on a LightCycler ™ LC24. Initial denaturation of target sequences was accomplished at 95°C for 1 min, followed by 28 cycles of denaturation at 95°C for 0 sec, annealing at 58°C for 15 sec, and elongation at 72°C for 5 sec. These assays are specific and can be performed on as little as 25 ng of total DNA or crude cell lysate a from fresh colony. It is thus possible to deter mine the genotype of B. anthracis strains in less than 1 hour.