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International Journal of Infectious Diseases: Volume 2, Number 2
Original Report: Rapid Diagnosis of Lyme Disease: Flagellin Gene-Based Nested Polymerase Chain Reaction for Identification of Causative Borrelia Species
Yukita Sato, DVM;* Tatsuya Konishi, MD;* Yoshio Hashimoto, MD; Hidetoshi Takahashi, MD; Kazuhiro Nakaya, DVM; Masahito Fukunaga, PhD;? and Minoru Nakao, PhD*

Int J Infect Dis 1997; 2(2): 64-73.

Objective: Each of Borrelia burgdorferi sensu stricto, Borrelia garinii, and Borrelia afzelii has characteristic restriction sites in its flagellin gene. The authors focused on this gene and developed a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis for rapid diagnosis of Lyme disease. Methods: External and internal primer sets were designed for nested PCR to amplify an approximately 580 bp fragment of the flagellin gene that includes species-specific restriction sites. DNA extracted from tissue samples of mice and humans were used as templates for PCR. The amplicons obtained were digested with HapII, HhaI, CelII, HincII, or DdeI endonuclease. Results: In mice experimentally infected with each of B. burgdorferi sensu stricto, B. garinii, and B. afzelii, borrelial DNA was detected irrespective of differences in the causative species. However, RFLP of the amplicons was able to identify the species. Skin biopsy samples from 11 Japanese patients with erythema migrans were subjected to both PCR and culture tests. Borrelial infections were detected in seven cases (64%) by PCR and eight cases (73%) by culture. All PCR-positive samples were also positive by culture. The causative species in human infections was easily identified as B. garinii by RFLP analysis of the amplicons. Conclusion: The nested PCR-RFLP system appears to be an easy and reliable diagnostic tool for the detection and species identification of borreliae in human cutaneous biopsies.

KEY WORDS: Borrelia, flagellin, Lyme disease, PCR, RFLP

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