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Increased Sensitivity and Specificity of Borrelia burgdorferi 16S Ribosomal DNA Detection

Sin Hang Lee MD, Veronica S. Vigliotti CMIAC, Jessica S. Vigliotti, William Jones, Suri Pappu MD
DOI: http://dx.doi.org/10.1309/AJCPI72YAXRHYHEE 569-576 First published online: 1 April 2010


The DNA of Borrelia burgdorferi spirochetes extracted by ammonium hydroxide was used as the template for nested polymerase chain reaction (PCR) amplification of the species-specific 16S ribosomal DNA (rDNA). The primers were those well known to be specific for signature sequence amplification of the B burgdorferi sensu lato 16S ribosomal RNA gene. The positive 293-base-pair nested PCR amplicon was subjected to routine direct automated Sanger sequencing. A 50-base sequence excised randomly from the sequencing electrophoretogram between the 2 nested PCR primer binding sites was sufficient for the Basic Local Alignment Search Tool (BLAST) analysis to validate the B burgdorferi sensu lato 16S rDNA without a reasonable doubt. Nested PCR increased the sensitivity of DNA detection by 100- to 1,000-fold. DNA sequence validation based on BLAST algorithms using the GenBank database practically eliminates any possibility of false-positive results due to molecular misidentification. This technology may be a valuable supplement to the current serologic tests for Lyme disease.

Key Words:
  • 16S rDNA
  • 16S rRNA gene
  • Borrelia burgdorferi sensu lato
  • Borrelia burgdorferi
  • Lyme disease
  • Nested polymerase chain reaction
  • DNA sequencing
  • Signature sequence
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