Detection of Borrelia burgdorferi in Human Blood and Urine Using the Polymerase Chain Reaction

Abstract

We investigated the use of the polymerase chain reaction (PCR) to detect Borrelia burgdorferi strain B-31 in human blood and urine experimentally inoculated with 5 and 1 borreliae/cm³, respectively, and to biotinylate a DNA probe specific for B. burgdorferi in the dot blot and Southern blot assays. When the blood and urine samples were subjected to PCR, a 370-bp amplified product was consistently visible on agarose gel electrophoresis after 30 and 45 cycles, respectively. The total human genomic DNA extracted from a 1-cm³ sample of inoculated blood was approximately 6.25 µg, and the total amount of B. burgdorferi DNA was estimated to be 0.01 pg/6.25 µg of the human DNA. For PCR, 2.5 µg of human DNA which contained the equivalent of 0.004 pg of borrelia DNA (approximately two borreliae) were used for enzymatic amplification. When 1/20 or 1/10 of the PCR-amplified products were used either for dot blot or Southern blot hybridization, the accessible copies of amplified B. burgdorferi DNA were sufficient for detectable hybridization to occur. PCR amplification of B. burgdorferi DNA in clinical specimens followed by dot blot hybridization may be a valuable adjunct or alternative to current but inadequate laboratory methods for the diagnosis of Lyme disease.

Evaluation of the detection ofBorrelia burgdorferi DNA in urine samples by polymerase chain reaction

“…However, these reports described either the in vitro testing of artificially contaminated urine samples [26] or the testing of a relatively small number of urine samples from patients [10,11,27,28], so that it seemed necessary to evaluate the PCR using a larger number of samples. Efficient methods for sample preparation are a prerequisite for PCR with urine, because urine can contain inhibitory substances which cannot be removed by some of the currently available protocols [29].…”

Methodological Aspects

“…Sensitivity in clinical samples can be lowered by losses dur- ing DNA preparation, by the presence of extraneous DNA (27,111), or by contamination with interfering and/or inhibitory substances (see the section on control of inhibition, below). One way to increase the sensitivity of PCR is by using a nested PCR procedure.…”

“…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).…”

Nachweis von Borrelien-DNA im Urin mittels Polymerase-Kettenreaktion in der rheumatologischen Labordiagnostik der Lyme-Borreliose

“…Zahlreiche Arbeitsgruppen konnten zeigen, daß bei Patienten mit gesicherter Lyme-Borreliose in kulturell sterilem Material wie Urin (3,12,14,16), Synovialflüs-sigkeit (5,7,14,20), Synovialmembran (6), Liquor (9. 10, 12, 14) oder Serum (14,16) Borrelia burgdorferi (Bb)-DNA nachgewiesen werden kann.…”