The Abbott m2000 system is a real time PCR amplification assay for the detection of Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (GC). The Abbott m2000sp automates the extraction of nucleic acids from each sample and the addition of master mix to the extracted nucleic acids. The 96-well plate is transferred to the m2000rt for real time PCR amplification using fluorescently labeled probes specific for CT and GC. The amount of fluorescence generated after each amplification cycle is measured and allows for the generation of a mean cycle number (CN) based on the cut-off controls. Specimens with a CN less than or equal to that number are considered positive for either CT or GC. This assay includes an internal inhibition control. The performance characteristics of this assay were established by Abbott and include male urethral, female endocervical, female vaginal swabs, and male or female urine. The performance characteristics for endocervical and urethral swabs placed in chlamydia transport medium (CTM) and either oral, vaginal, or rectal swabs collected and placed into a dry specimen sheath were established in our laboratory. All other sample types are unacceptable. The limit of detection is less than 1 IFU (infection forming units) per assay for CT and less than 1 CFU (colony forming units) per assay for GC.
Roche COBAS 4800
The cobas 4800 CT/GC assay is based on two major processes; specimen processing to elute CT/GC DNA is automated on the x480 instrument and amplification of CT/GC DNA using real-time PCR on the z480 instrument. Specimens are digested and lysed under denaturing conditions with the resulting DNA being purified through absorption onto magnetic glass particles. The DNA is eluted from the glass particles and transferred to a microwell plate containing DNA amplification master mix. The plate is sealed and moved to the real-time instrument, where it undergoes PCR amplification. Fluorescently labeled reporter probes specific for CT and GC are bound to their complementary DNA sequence and fluoresce at a specific wavelength when cleaved by nuclease activity during each amplification cycle. The fluorescent intensity is measured at the end of each amplification cycle and can be used to determine the presence or absence of CT or GC DNA in the specimen. This assay includes an internal inhibition control. The performance characteristics of this assay were established in this laboratory for cervical, patient or clinician obtained vaginal swabs, urethral swabs, oral or rectal swabs and either male or female urine specimens. All other sample types are unacceptable. The limit of detection is 1-10 IFU per ml for CT and 2.25-100 CFU per ml for GC.
Hologic (GenProbe Aptima Combo 2)
The Hologic Aptima Combo 2 assay combines target capture of rRNA, amplification of the captured target by transcription mediated amplification, and detection of the amplified target through the use of dual kinetic chemiluminescent probes specific for either CT or GC. Target rRNA is released from the specimen and captured on magnetic microparticles containing analyte specific capture oligomers. Enzymatic target amplification occurs through DNA intermediaries. The labeled DNA probes specific for CT or GC hybridize to the amplified target RNA to form stable RNA:DNA hybrids. The light emitted from these stable hybrids is measurable and reported as relative light units (RLU). Detection of CT or GC is based on total RLU and the unique kinetic profile produced by each probe. The performance characteristics of this assay were established by Hologic and include endocervical, urethral, vaginal and urine specimens. The performance characteristics for rectal and oral swabs were established in our laboratory. All other sample types are unacceptable. The limit of detection is 1 IFU per assay for CT and 50 cells per assay for GC.
Becton Dickinson Viper
The Becton Dickinson (BD) Qx CT/GC assay uses strand displacement amplification for the direct detection of CT and GC. Nucleic acid is extracted from each sample using high pH, with the liberated DNA being bound to metal beads, washed, and eluted for amplification. The isolated DNA is transferred to a priming microwell, which contains amplification primers and fluorescently labeled detector probes. After incubation, the mixture is transferred to the amplification microwell, which contains DNA polymerase and a restriction endonuclease, necessary components of the strand displacement assay. The amplification wells are incubated in a thermally controlled fluorescent reader, with the presence or absence of CT or GC determined by calculating the peak fluorescence over the course of the amplification and by comparison to a predetermined threshold value. An extraction control (EC) is incorporated into each reaction to confirm that proper extraction has occurred. This process is completely automated on the Viper instrument in extracted mode. The performance characteristics of this assay were established by Becton Dickinson and include endocervical and male urethral swabs, patient or clinician obtained vaginal swabs, and urine collected using the Qx transport kits. All other sample types are unacceptable. The limit of detection is less than 30 IFU for CT and less than 100 cells per assay for GC.