title: A real-time quantitative polymerase chain reaction assay for the detection of Chlamydia in the mouse genital tract model.
abstract: Chlamydia trachomatis is a human pathogen that infects genital tracts in women. Disease control may be achieved through development of an efficacious vaccine. A mouse genital tract model serves as a tool for evaluation of vaccine candidates. Currently, assessment of infection in mice is performed by enumeration of inclusion-forming units (IFUs) through microscopic counting of fluorescently stained bacteria. We have developed a highly sensitive real-time quantitative polymerase chain reaction (RT-qPCR) assay for enumeration of Chlamydia from mouse genital tracts to increase assay sensitivity, remove subjectivity, and improve sample throughput. The qPCR assay uses a 16S ribosomal gene sequence that is conserved across Chlamydia species and serovars, resulting in detection of multiple serovars of C. trachomatis, as well as Chlamydia muridarum and Chlamydia pneumoniae. The PCR assay provided results similar to IFU enumeration (94% agreement between the 2 assays) and is highly sensitive and specific with less inherent subjectivity than traditional enumeration methods.
authors.affiliation: Vaccine Basic Research, Merck Research Laboratories, West Point, PA 19486-0004, USA. melissa_wooters@merck.com
authors.forename: Melissa A
authors.initials: MA
authors.lastname: Wooters
authors.forename: Robin M
authors.initials: RM
authors.lastname: Kaufhold
authors.forename: Jodie A
authors.initials: JA
authors.lastname: Field
authors.forename: Lani
authors.initials: L
authors.lastname: Indrawati
authors.forename: Jon H
authors.initials: JH
authors.lastname: Heinrichs
authors.forename: Jeffrey G
authors.initials: JG
authors.lastname: Smith
mesh_terms: D000818:Animals; D015373:Bacterial Typing Techniques / Q000379:methods*; D002460:Cell Line; D002689:Chlamydia / Q000235:genetics / Q000302:isolation & purification*; D002690:Chlamydia Infections / Q000382:microbiology*; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004195:Disease Models, Animal; D005260:Female; D005836:Genitalia, Female / Q000382:microbiology*; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D016133:Polymerase Chain Reaction / Q000379:methods*; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014621:Vagina / Q000382:microbiology
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S