USE OF DNA PURIFICATION KITS FOR POLYMERASE-CHAIN-REACTION TESTING OFGEN-PROBE CHLAMYDIA-TRACHOMATIS PACE-2 SPECIMENS

Background and Objectives: Confirmation testing using nucleic acid amp lification has been shown to improve the sensitivity and specificity o f screening tests for Chlamydia trachomatis. However, no critical info rmation on the use of these techniques as an adjunct to Gen-Probe hybr idization testing, one of the most common screening methods, has been reported to date. We examined the Roche AMPLICOR PCR C. trachomatis Te st (Roche Diagnostic Systems, Branchburg, NJ) as a confirmatory test f or the Gen-Probe PACE 2 C. trachomatis Test (San Diego, CA). Further, to mitigate the possible effect of interfering compounds in the Gen-Pr obe PACE 2 transport medium, we tested various DNA purification techni ques. Study Design: C. trachomatis elementary bodies were used to spik e PACE 2 Transport medium, which was serially diluted, then tested by polymerase chain reaction (PCR). Six parallel dilution series were con ducted: (1) saline dilutions tested by the Syva Direct Specimen Test, (2) Roche AMPLICOR transport medium dilutions tested by PCR, and (3-6) dilutions in PACE 2 transport medium purified respectively by GENECLE AN II (BIO101, Vista, CA), Puregene (Gentra Systems, Inc., Research Tr iangle Park, NC), Microcon 100 (Amicon, Inc., Beverly, MA) DNA isolati on kits, and no DNA purification, all tested by PCR. The system giving the best results by in vitro endpoint dilution trials was then used t o confirm human specimens previously tested by the Gen-Probe method. R esults: PCR detected C. trachomatis at 11 twofold dilutions greater th an PACE 2 and equivalent to detection of a single elementary body by S yva Direct Specimen Test. DNA purification of spiked PACE 2 transport medium by the Microcon 100 kit produced the most consistent PCR detect ion endpoints, equivalent to endpoints of spiked AMPLICOR transport me dium. Endpoints with no DNA purification step were variable and lower. Of 78 endocervical specimens negative by PACE 2 and Gen-Probe Probe C ompetition Assay, 12 (15.3%) were positive by Microcon DNA purificatio n/PCR testing. Conclusions: PCR can be used as confirmation method for Gen-Probe PACE 2 testing, but testing must be performed with a DNA pu rification procedure.