Detection of Borrelia burgdorferi in biological samples using the polymerase chain reaction assay

Detection of Borrelia burgdorferi in skin biopsies from patients with morphea by polymerase chain reaction

“…Cultural isolation on Barbour-Stoenner-Kclly medium provides certain evidence of the presence of Lyme-disease agent in the tissue [29]. A recent biotechnological method, the amplification of DNA with PCR seems to open new prospects for the detection of Borreliy genome in tissues, particularly when these spirochetes are present in a small numbers [30,31]. A recent biotechnological method, the amplification of DNA with PCR seems to open new prospects for the detection of Borreliy genome in tissues, particularly when these spirochetes are present in a small numbers [30,31].…”

“…Unfortunately, the num-ber of false-negatives is high: the detection of B. burgdorferi is very difficult owing to the scarceness of microorganisms in the tissue and B. burgdorferi is a delicate germ which grows with difficulty in the culture medium. A recent biotechnological method, the amplification of DNA with PCR seems to open new prospects for the detection of Borreliy genome in tissues, particularly when these spirochetes are present in a small numbers [30,31]. Ranki et al [21] and Wienecke ct al.…”

The Role of Culture and Nucleic Acid Amplification in Diagnosis of Lyme Borreliosis

“…Of these, skin biopsy samples taken from patients with EM were the most frequently tested specimens Wienecke et al, 1993;Muellegger et al, 1995Muellegger et al, , 1996von Stedingk et al, 1995;Picken et al, 1997;Rijpkema et al, 1997;Brettschneider et al, 1998;Oksi et al, 2001;Liveris et al, 2002;Wang, 2002). Other commonly used clinical specimens for PCR analysis include whole blood (Liebling et al, 1993), serum (Wallach et al, 1993;Demaerschalck et al, 1995) or plasma specimens (Goodman et al, 1995;Oksi et al, 2001), cerebrospinal fluid from patients with neuroborreliosis (Debue et al, 1991;Keller et al, 1992;Luft et al, 1992;Liebling et al, 1993;Pachner and Delaney, 1993;Zbinden et al, 1994;Christen et al, 1995;Nocton et al, 1996;Priem et al, 1997;Schwaiger et al, 2001;Ornstein et al, 2002), and synovial fluid/tissue from patients with Lyme arthritis (Debue et al, 1991;Liebling et al, 1993;Bradley et al, 1994;Persing et al, 1994;Jaulhac et al, 1996;Nocton et al, 1996;Priem et al, 1997;Eiffert et al, 1998;van der Heijden et al, 1999;Schwaiger et al, 2001;Schnarr et al, 2001). One study suggested that plasma may be the blood component most efficient for PCR analysis (Goodman et al, 1995)....…”

Methodological Aspects

“…Most researchers in the United States have used the genes coding for the outer surface proteins, the ospA and ospB genes, which are located on a linear plasmid (Table 1). These genes are known to be highly variable (191) and have been found not to amplify all strains isolated in Europe (31,84,122,137,156). Although more than 20 different gene segments were amplified, only a few direct comparisons have been published.…”

“…Not surprisingly, procedures involving simpler methods have been developed; these include simple boiling of the specimen (88), centrifugation and boiling (31), alkali lysis (111), adsorption to coated or uncoated silica in the presence of chaotropic salts (7,21,51,68,105), boiling and concentration by centrifugation and ultrafiltration (128), and boiling in the presence of a cation exchanger (Chelex 100; Bio-Rad, Richmond, Calif.) (93). Although the mechanism by which Chelex improves the DNA extraction, is not known, it is thought to aid in stabilizing the DNA double helix (182).…”