DNA sequencing validation of Chlamydia trachomatis and Neisseria gonorrhoeae nucleic acid tests

Am J Clin Pathol. 2008 Jun;129(6):852-9. doi: 10.1309/Y2202RVCGRWW1RWB.


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.

Publication types

  • Validation Study

MeSH terms

  • Base Sequence
  • Chlamydia Infections / diagnosis*
  • Chlamydia Infections / microbiology
  • Chlamydia trachomatis / genetics*
  • Chlamydia trachomatis / isolation & purification
  • DNA, Bacterial / analysis*
  • False Negative Reactions
  • Gonorrhea / diagnosis*
  • Gonorrhea / microbiology
  • Humans
  • Molecular Diagnostic Techniques
  • Molecular Sequence Data
  • Neisseria gonorrhoeae / genetics*
  • Neisseria gonorrhoeae / isolation & purification
  • Nucleic Acid Amplification Techniques*
  • Predictive Value of Tests
  • Reagent Kits, Diagnostic
  • Sequence Analysis, DNA


  • DNA, Bacterial
  • Reagent Kits, Diagnostic