title: RNA amplification by nucleic acid sequence-based amplification with an internal standard enables reliable detection of Chlamydia trachomatis in cervical scrapings and urine samples.
abstract: In the present study, the suitability of RNA amplification by nucleic acid sequence-based amplification (NASBA) for the detection of Chlamydia trachomatis infection was investigated. When comparing different primer sets for their sensitivities in NASBA, use of both the plasmid and omp1 targets resulted in a detection limit of 1 inclusion-forming unit (IFU), while the 16S rRNA appeared to be the most sensitive RNA target for amplification (10(-3) IFU). In contrast, for DNA amplification by PCR, the plasmid target was optimal (10(-2) IFU), which is 10 times less sensitive than rRNA NASBA. To exclude false negativity in NASBA detection because of inhibition of amplification and/or inefficient sample preparation, an internal standard was developed. The internal control was added prior to sample preparation. This 16S rRNA NASBA with an internal control was compared with a plasmid DNA PCR by using a group of C. trachomatis-negative (n = 41) and -positive (n = 37) cervical scrapings, as determined by enzyme immunoassay (EIA). In addition, urine samples from the EIA-positive women were tested (n = 17). Both NASBA and PCR assays were able to detect C. trachomatis in all EIA-positive cervical scrapings, the corresponding urine samples, and two samples from the EIA-negative group. The internal NASBA standard was found clearly in all EIA-negative samples. In conclusion, these results indicate that detection of C. trachomatis by RNA amplification by NASBA with an internal standard is a suitable and highly sensitive detection method, with potential use in the diagnosis of urogenital C. trachomatis infections with cervical scrapings as well as urine specimens.
authors.affiliation: Department of Pathology, University Hospital Vrije Universiteit, Amsterdam, The Netherlands.
authors.forename: S A
authors.initials: SA
authors.lastname: Morré
authors.forename: P
authors.initials: P
authors.lastname: Sillekens
authors.forename: M V
authors.initials: MV
authors.lastname: Jacobs
authors.forename: P
authors.initials: P
authors.lastname: van Aarle
authors.forename: S
authors.initials: S
authors.lastname: de Blok
authors.forename: B
authors.initials: B
authors.lastname: van Gemen
authors.forename: J M
authors.initials: JM
authors.lastname: Walboomers
authors.forename: C J
authors.initials: CJ
authors.lastname: Meijer
authors.forename: A J
authors.initials: AJ
authors.lastname: van den Brule
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000235:genetics; D001431:Bacteriological Techniques / Q000592:standards / Q000706:statistics & numerical data; D001483:Base Sequence; D002584:Cervix Uteri / Q000382:microbiology*; D002690:Chlamydia Infections / Q000175:diagnosis / Q000382:microbiology; D002692:Chlamydia trachomatis / Q000235:genetics* / Q000302:isolation & purification*; D017931:DNA Primers / Q000235:genetics; D005069:Evaluation Studies as Topic; D005260:Female; D005798:Genes, Bacterial; D006801:Humans; D021141:Nucleic Acid Amplification Techniques; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods / Q000706:statistics & numerical data; D012329:RNA, Bacterial / Q000235:genetics* / Q000302:isolation & purification*; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012015:Reference Standards; D012680:Sensitivity and Specificity; D014556:Urine / Q000382:microbiology*; D002577:Uterine Cervical Diseases / Q000175:diagnosis / Q000382:microbiology
chemical_list: D001425:Bacterial Outer Membrane Proteins; D017931:DNA Primers; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S