Detection and quantification of Lyme spirochetes using sensitive and specific molecular beacon probes

We show here that molecular beacons are effective, sensitive and specific probes for detecting and estimating wide-ranging numbers of B. burgdorferi in the presence of mouse DNA. In our assays, the spirochete recA and the mouse nidogen gene amplicons were detected simultaneously using molecular beacons labeled with different fluorophores. We further validated the application of these probes by quantifying the wild-type strain and bgp-defective mutant of B. burgdorferi. The bgp-defective mutant shows a ten-fold reduction in the level of spirochetes present in various tissues.The high sensitivity and specificity of molecular beacons makes them superior probes for the detection of small numbers of B. burgdorferi. Furthermore, the use of molecular beacons can be expanded for the simultaneous detection and quantification of multiple pathogens in the infected hosts, including humans, and in the arthropod vectors.Lyme disease, caused by the spirochete Borrelia burgdorferi, is a highly prevalent multisystemic illness that affects the heart, joints, skin, musculoskeletal and nervous system. Persistent infection with the spirochete results in potentially severe manifestations, such as, carditis, arthritis, acrodermatitis chronicum atrophicans and neuroborreliosis. The severity of the Lyme disease depends on several factors including; genotypes of both the host and the infecting Borrelia strain, age of the host, simultaneous infection with another tick-transmitted pathogen and the spirochete burden in the infected tissue [1-7].The B. burgdorferi genome is relatively small (1.52 Mb) in size. Although the spirochete lacks major biosynthetic pathways, it contains a large number of surface proteins. Several of these are adhesins, which mediate attachment to various cell lines [8-13]. Each adhesin could contribute to the tissue specific colonization by the spirochetes. Alternatively, the presence of multiple adhesins exhibiting specificity for the same receptor can create a redunda