title: DNA sequencing validation of Chlamydia trachomatis and Neisseria gonorrhoeae nucleic acid tests.
abstract: DNA sequencing was used to confirm Chlamydia trachomatis and Neisseria gonorrhoeae nucleic acids in endocervical swab samples. DNA in residues of the samples with positive results by 2 commercial kits was subjected to nested polymerase chain reaction (PCR) amplification. The nested PCR amplicons were used as templates for direct automated DNA sequencing. A 40-base signature sequence was sufficient to achieve unequivocal validation of C trachomatis cryptic plasmid and gonococcal opa gene DNA. DNA with a signature sequence specific for C trachomatis was identified in all 14 samples and for N gonorrhoeae in all 13 samples with positive results by the commercial kits for these 2 microbes. In a low-prevalence population, PCR retesting of 289 samples with initial negative results by a non-nucleic acid amplification assay revealed 3 samples positive for C trachomatis and 2 samples positive for N gonorrhoeae that were missed by the commercial kit. DNA sequencing is a useful tool in validating molecular diagnostics.
authors.affiliation: Department of Pathology, Milford Hospital, Milford, CT.
authors.forename: Sin Hang
authors.initials: SH
authors.lastname: Lee
authors.forename: Veronica S
authors.initials: VS
authors.lastname: Vigliotti
authors.forename: Suri
authors.initials: S
authors.lastname: Pappu
mesh_terms: D001483:Base Sequence; D002690:Chlamydia Infections / Q000175:diagnosis* / Q000382:microbiology; D002692:Chlamydia trachomatis / Q000235:genetics* / Q000302:isolation & purification; D004269:DNA, Bacterial / Q000032:analysis*; D005188:False Negative Reactions; D006069:Gonorrhea / Q000175:diagnosis* / Q000382:microbiology; D006801:Humans; D025202:Molecular Diagnostic Techniques; D008969:Molecular Sequence Data; D009344:Neisseria gonorrhoeae / Q000235:genetics* / Q000302:isolation & purification; D021141:Nucleic Acid Amplification Techniques; D011237:Predictive Value of Tests; D011933:Reagent Kits, Diagnostic; D017422:Sequence Analysis, DNA
chemical_list: D004269:DNA, Bacterial; D011933:Reagent Kits, Diagnostic