A Reverse Transcriptase–Polymerase Chain Reaction Assay of Borrelia burgdorferi 16S rRNA for Highly Sensitive Quantification of Pathogen Load in a Vector

We developed a real-time quantitative detection assay for the pathogen Borrelia burgdorferi, a Lyme borreliosis(LB) agent, using reverse transcription–polymerase chain reaction (RT-PCR) with primers and probe for a Borrelia genus-specific region of 16S ribosomal RNA. The standard curve of the assay was linear by semi-log plot over more than five orders of magnitude, and the detection limit of the assay was one thousandth of a single cell of B. burgdorferi. The minimum target level for detection using the RT-PCR assay for 16S RNA was 40-fold lower than the RT-PCR assay for messenger RNA of ospA, a highly expressed, plasmid-borne gene, and 1600-fold lower than the RT-PCR assay for messenger RNA of p66, a chromosome-borne gene of B. burgdorferi. The 16S rRNA assay was then applied in an experimental setting for monitoring the spirochetal load in B. burgdorferi-infected Ixodes scapularis ticks before and after they fed on Peromyscus leucopus mice immunized with recombinant OspA. Unfed infected ticks had a mean of 2,240 spirochetes per tick, and after feeding on non-immunized mice and engorgement, the mean number of spirochetes increased to 223,900 per tick. In contrast, there were either no or ≤7 spirochetes in ticks that had fed on OspA-immunized mice.