Purpose/Principle

In this assay, DNA extracted from patient samples is subjected to real-time PCR amplification using primers and fluorescent probes directed against three target sequences in the Spn genome: lytA, piaB, and SP2020 (5-7). The product is detected on an Applied Biosystems 7500 Taqman™ instrument using 5’-nuclease methodology and quantitated by extrapolation to a standard curve of log10 dilutions of commercially available quantified Spn whole organism. The assay is linear across five orders of magnitude and can detect as few as 100 copies/mL. An exogenous positive control with its own set of primers and probes is co-amplified in each reaction to determine extraction efficiency and detect inhibition. Results are reported as number of copies of Spn per mL of fluid sample.