TaqMan-Based Detection of Trichomonas vaginalis DNA from Female Genital Specimens

摘要

A double-labeled fluorescent probe was designed and evaluated for detecting Trichomonas vaginalis DNA in a 5′ nuclease (TaqMan) assay. The T. vaginalis-specific probe contains a 5′-fluorescein (5′-FAM) and a 3′-rhodamine (TAMRA) derivative. Female genital secretions were collected on Amplicor (Roche Molecular, Indianapolis, Ind.) swabs and by a transport system used forChlamydia trachomatis and/or Neisseria gonorrhoeae DNA detection by PCR. Five hundred fifty-two female genital specimens, of which 248 (45%) were vaginal specimens and 304 (55%) were introital, were tested for both T. vaginalisDNA and viable microorganisms using the 5′ nuclease assay and broth culture, respectively. Of these, 304 of 552 (55%) were also evaluated by direct microscopic examination for the characteristic motile organism. After resolving discrepancies, the comparisons produced an analytical sensitivity and specificity for the TaqMan-based PCR assay of 97.8 and 97.4%, respectively. As a result, ΔRQ values (differences in fluorescence due to probe hybridization and resulting 5′-FAM cleavage from the specific PCR product) of ≥2.0 and ≤1.5 were established for T. vaginalis-positive and -negative cutoffs, respectively. ΔRQ values between 1.5 and 2.0 were considered indeterminate. Overall findings revealed a high level of agreement between PCR and culture for detecting T. vaginalis. Potential benefits of the 5′ nuclease assay include a greater sensitivity compared to direct microscopic examination and the ease of testing large numbers of clinical specimens in a significantly shorter turnaround time compared to culture.