PCR-reverse line blot typing method underscores the genomic heterogeneity of Borrelia valaisiana species and suggests its potential involvement in Lyme disease

Abstract

Detection of the Borrelia burgdorferi sensu lato complex in biological samples is currently done by conventional immunological and molecular biological methods. To improve on the accuracy of these methods and to simplify the procedure for testing large numbers of samples, a solid-phase sandwich hybridization system readily applicable to the detection of PCR products has been designed. This colorimetric detection system relies on the use of polybiotinylated detection probes and of specific capture oligonucleotides covalently linked at allocated positions on nylon membrane strips. From a phylogenetic analysis on a great number of ospA gene sequences, we have designed and synthesized a set of PCR primers specific to the five Borrelia burgdorferi sensu lato genospecies present in Europe and a subset of probes (capture and detection probes) specific to these five genospecies (B. burgdorferi sensu stricto, B. garinii, B. afzelii, B. valaisiana, and B. lusitaniae). This combined PCR hybridization system was evaluated with a large number of various B. burgdorferi isolates and clinical specimens. These analyses clearly showed that the system could be used as a typing method to distinguish five genospecies belonging to the B. burgdorferi sensu lato complex. In addition, the study showed that B. valaisiana strains might be more heterologous than suspected up to now and clustered into three genomic groups.

FIG. 1.

Individual hybridization of about 10¹² copies of PCR-amplified DNA fragments specific to B. burgdorferi sensu stricto (B31), B. garinii (ZQ1), B. afzelii (VS461), B. valaisiana (VS116), and B. lusitaniae (PotiB1) species (strips 2 to 6, respectively). Nylon membrane strip 1 represents the negative control; unrelated DNA-amplified product was mixed with the hybridization buffer. I, II, III, IV, and V indicate the positions of the capture probes specific to B. burgdorferi sensu stricto, B. garinii, B. afzelii, B. valaisiana, and B. lusitaniae, respectively.

FIG. 2.

Phylogenetic tree of ospA DNA sequences from 40 isolates, including some ospA sequences (sequences 49, 219, 228, 229, 248, 249, 889, and 901 and that from patient 6) obtained in this study (in boldface type and underscored).