Sin Hang Lee, MD; Veronica S. Vigliotti, CMIAC; Jessica S. Vigliotti; William Jones; Suri Pappu, MD
Am J Clin Pathol (2015) 133 (4): 569-576. DOI: https://doi.org/10.1309/AJCPI72YAXRHYHEE
Published: 01 August 2015
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Several polymerase chain reaction (PCR)-based nucleic acid tests have been introduced for the detection of B burgdorferi DNA in clinical specimens as an adjunct to the serologic assays, including the DNA tests for chromosomally carried genes such as ribosomal RNA (rRNA) genes, flaB, recA, and p66, and the plasmid-carried ospA.5 In general, the sensitivity of these PCR tests for the detection of B burgdorferi DNA in blood, plasma, or serum samples from patients with Lyme disease is low, ranging from 0% to 59%,6–8 with an 18% mean detection sensitivity in the United States.9 The sensitivity for PCR detection of Lyme spirochete DNA in synovial fluid samples seems to be higher.10 False-positive PCR test results can sway physicians to making erroneous diagnoses of Lyme disease, which may lead to inappropriate treatment with potentially serious complications.11,12
Because DNA sequences of the 16S rRNA gene, or the 16S rDNA, of all bacteria contain their respective unique hypervariable regions, a proposal has been made to select certain segments of these hypervariable regions as species-specific or type-specific signature sequences for bacterial identification in clinical microbiology.13,14 PCR primers designed to amplify regions of the 16S rRNA gene have been used to distinguish various groups of B burgdorferi.15 However, this gene is also known to contain highly conserved regions that are shared among the various isolates of B burgdorferi and non-Lyme Borrelia.16 A new primer designated as TEC117 has been introduced to pair with a previously reported LD2 primer15 to improve the specificity of the 16S rDNA–based PCR, which would allow differentiation of B burgdorferi sensu lato from other Borrelia species that do not cause Lyme disease.
In this article, we report a practical laboratory procedure by combining these 2 sets of primers to develop a nested (heminested) PCR assay for detection of Lyme Borrelia DNA in human body fluids and engorged Ixodes insects removed from skin bites. A target 293-base-pair (bp) nested PCR amplicon was further subjected to direct automated DNA sequencing to validate that the amplicon indeed consists of a signature DNA sequence of the B burgdorferi sensu lato 16S rRNA gene. Similar procedures have been used for human papillomavirus genotyping18–21 and for molecular testing of Chlamydia trachomatis and Neisseria gonorrhoeae.21,22
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