Disclosed are oligonucleotides useful in methods for determining whether a sample contains Borrelia afzelii, a spirochete which is a causative agent of Lyme disease in humans. These oligonucleotides, which have nucleotide sequences derived from a coding segment of the gene encoding the p24 gene for the outer surface protein of Borrelia afzelii, are useful as forward and reverse primers for a polymerase chain reaction using nucleic acids from a biological sample as templates, and as probes for detecting any resultant amplicon. Detection of an amplicon indicates the sample contains Borrelia afzelii. Real-time PCR and detection using florescence resonance energy transfer is disclosed.