Species-specific plasmid sequences for PCR identification of the three species of Borrelia burgdorferi sensu lato involved in Lyme disease

Abstract

Species-specific sequences were shown to be carried by plasmids of the three main species of Borrelia burgdorferi sensu lato involved in Lyme disease. Libraries of the 16-, 33-, and 25-kb plasmids of B. burgdorferi sensu stricto, Borrelia garinii, and Borrelia afzelii, respectively, were then built and used to isolate species-specific sequences. After sequencing of the cloned inserts, three sets of primers were designed. They were shown to determine species-specific PCR amplification products. The sensitivities of the PCR assay with these primers were 100 spirochetes for B. burgdorferi sensu stricto and 1,000 spirochetes for B. garinii and B. afzelii. The usefulness of these primers for the identification of species in biological samples (tick, serum, and cerebrospinal fluid samples) was ascertained.

FIG. 1

Hybridization of the 16-kb plasmid of B. burgdorferi sensu stricto, the 33-kb plasmid of B. garinii, and the 25-kb plasmid of B. afzelii with DNAs of the three species of B. burgdorferi sensu lato involved in Lyme disease. DNAs (100 ng) of the B31 (dot 1), IP1 (dot 2), IP2 (dot 3), IP3 (dot 4), and IRS (dot 5) strains of B. burgdorferi sensu stricto, the 20047 (dot 6), N34 (dot 7), G25 (dot 8), and P/Bi (dot 9) strains of B. garinii, and the VS461 (dot 10), UO1 (dot 11), UMO1 (dot 12), and Iper3 (dot 13) strains of B. afzelii were denatured and spotted onto a nylon membrane. The membrane was incubated in the presence of the 16-kb radiolabeled plasmid of B. burgdorferi sensu stricto IP1 (A), the 33-kb radiolabeled plasmid of B. garinii N34 (B), or the 25-kb radiolabeled plasmid of B. afzelii UO1 (C). The membrane was autoradiographed.

FIG. 2

Specificity of the chosen primers for amplification of B. burgdorferi sensu stricto, B. garinii, or B. afzelii DNA. DNAs from B. burgdorferi sensu stricto B31 (lanes 3), IP1 (lanes 4), and IRS (lanes 5), B. garinii 20047 (lanes 6), N34 (lanes 7), and G25 (lanes 8), B. afzelii VS461 (lanes 9), UO1 (lanes 10), and Iper3 (lanes 11), B. japonica Cow611C (lanes 12), B. hermsii (lanes 13), B. parkeri (lanes 14), and B. turicatae (lanes 15) were amplified with the MC16 (A), MC33 (B), or MC25 (C) primers. The amplified products were separated on a 1.5% agarose gel and revealed under UV light after the addition of ethidium bromide (10 μg/ml). Lanes 2, control without DNA; lanes 1, DNA molecular mass markers (587, 540, 504, 458, 434, 267, 234, 213, 192, 184, and 124 bp).

FIG. 3

Sensitivity of the B. burgdorferi sensu stricto, B. garinii, and B. afzelii species-specific primers. DNA corresponding to 10⁷ (lanes 2), 10⁶ (lanes 3), 10⁵ (lane 4), 10⁴ (lanes 5), 10³ (lanes 6), 10² (lanes 7), 10 (lanes 8), 1 (lanes 9), or 0 (lanes 10) spirochetes of B. burgdorferi sensu stricto IP1 (A), B. garinii N34 (B), or B. afzelii UO1 (C) was amplified with the MC16 (A), MC33 (B), or MC25 (C) primers. The amplified products were electrophoretically separated in a 1.5% agarose gel and revealed under UV light after the addition of ethidium bromide (10 μg/ml). Lanes 1, DNA molecular mass markers (587, 540, 504, 458, 434, 267, 234, 213, 192, 184, and 124 bp).