Chlamydia DNA extraction for use in PCR: stability and sensitivity in detection.
We evaluated multiple procedures for extracting chlamydial DNA from specimens for detection in PCR tests. Commercial kits and an in-house method were tested for their sensitivity and utility. Quantifiable chlamydial elementary bodies (EB) were used for spiking buffy coats from EDTA-collected blood. EBs of Chlamydia pneumoniae at 2,500 and 25 EB/ml were used as specimens for DNA extraction using seven different procedures. These included either columns (3 procedures), centrifugation (1), glass (1), or patented extraction matrices (2), coupled with either alcohol precipitation (6) or heat-detergent treatment (1). Five procedures required 10-40 minutes manipulation; two required 2-5 hours. PCR results for DNA extracts using chlamydial 16S genus primers were generally more intensely positive with denser bands on electrophoresis gels for the higher concentrations of EB (up to 4+ for stained product on gels) than was PCR with lower EB concentrations (up to 2+). Further, the incidence of procedures with positive results was: 5 of 7 for chlamydial genus primers with 5 EB vs. 6 of 7 with 500 EB. Maximal sensitivity for one of the extractions was in the range of 2.5-5.0 EB/ml of test specimen with 4 of 5 replicates being positive with EB controls or extracts. Extracts were stable up to 2+ weeks at 4 degrees C and were effective in multiplexing with fluorescent-tagged primers. Taking into consideration the time factor and sensitivities, the two procedures with extraction matrices are favored for routine laboratory use.
Daugharty
H
H
National Center for Infectious Diseases, Centers for Disease Control, Atlanta, Georgia 30333, USA. had1@cdc.gov
Skelton
S K
SK
Messmer
T
T
D002689:Chlamydia / Q000235:genetics*; D004269:DNA, Bacterial / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity
D004269:DNA, Bacterial