Increased Sensitivity and Specificity of Borrelia burgdorferi 16S Ribosomal DNA

Excerpt:

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.