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DNA Sequencing Validation of Chlamydia trachomatis and Neisseria gonorrhoeae Nucleic Acid Tests

Sin Hang Lee MD, Veronica S. Vigliotti CMIAC, Suri Pappu MD
DOI: http://dx.doi.org/10.1309/Y2202RVCGRWW1RWB 852-859 First published online: 1 June 2008


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.

Key Words:
  • DNA sequencing validation
  • Neisseria gonorrhoeae DNA
  • Chlamydia trachomatis DNA
  • Cryptic plasmid DNA
  • Gonococcal opa genes
  • Nucleic acid tests