TaqMan-based detection of Trichomonas vaginalis DNA from female genital specimens.
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 for Chlamydia 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. vaginalis DNA 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, DeltaRQ values (differences in fluorescence due to probe hybridization and resulting 5'-FAM cleavage from the specific PCR product) of > or =2.0 and < or =1.5 were established for T. vaginalis-positive and -negative cutoffs, respectively. DeltaRQ 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.
Jordan
J A
JA
Magee-Women's Research Institute, University of Pittsburgh, Pittsburgh, Pennsylvania 15213, USA. jordanja+@pitt.edu
Lowery
D
D
Trucco
M
M
D000818:Animals; D003470:Culture Media; D015342:DNA Probes; D016054:DNA, Protozoan / Q000032:analysis*; D005260:Female; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D011247:Pregnancy; D012680:Sensitivity and Specificity; D019914:Taq Polymerase / Q000378:metabolism*; D014247:Trichomonas Vaginitis / Q000175:diagnosis* / Q000382:microbiology; D014246:Trichomonas vaginalis / Q000235:genetics / Q000302:isolation & purification*; D014621:Vagina / Q000469:parasitology*
D003470:Culture Media; D015342:DNA Probes; D016054:DNA, Protozoan; D019914:Taq Polymerase