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 <or=7 spirochetes in ticks that had fed on OspA-immunized mice.
Ornstein
Katharina
K
Clinical and Experimental Infectious Medicine Section, Department of Clinical Sciences, Lund University, Lund, Sweden. katharina.ornstein@med.lu.se
Barbour
Alan G
AG
D000818:Animals; D000954:Antigens, Surface / Q000235:genetics / Q000276:immunology; D001096:Arachnid Vectors / Q000382:microbiology*; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000276:immunology; D001426:Bacterial Proteins / Q000235:genetics; D001428:Bacterial Vaccines / Q000235:genetics / Q000276:immunology; D025065:Borrelia burgdorferi / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D017931:DNA Primers / Q000737:chemistry; D005260:Female; D018884:Ixodes / Q000382:microbiology*; D008074:Lipoproteins / Q000235:genetics / Q000276:immunology; D008193:Lyme Disease / Q000175:diagnosis / Q000517:prevention & control / Q000635:transmission; D010542:Peromyscus / Q000276:immunology / Q000382:microbiology; D018272:Porins / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis; D012336:RNA, Ribosomal, 16S / Q000032:analysis* / Q000235:genetics; D012044:Regression Analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013223:Statistics as Topic; D017282:Tick-Borne Diseases / Q000635:transmission
D000954:Antigens, Surface; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D001428:Bacterial Vaccines; D017931:DNA Primers; D008074:Lipoproteins; C107893:Oms66 protein, Borrelia; C072045:OspA protein; D018272:Porins; D012333:RNA, Messenger; D012336:RNA, Ribosomal, 16S