Increased sensitivity and specificity of Borrelia burgdorferi 16S ribosomal DNA detection.

Abstract:

: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.

journal_name

Am J Clin Pathol

authors

Lee SH,Vigliotti VS,Vigliotti JS,Jones W,Pappu S

doi

10.1309/AJCPI72YAXRHYHEE

subject

Has Abstract

pub_date

2010-04-01 00:00:00

pages

569-76

issue

4

eissn

0002-9173

issn

1943-7722

pii

133/4/569

journal_volume

133

pub_type

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