An optimized PCR leads to rapid and highly sensitive detection of Borrelia burgdorferi in patients with Lyme borreliosis

Abstract

The present study aimed at developing an optimized PCR protocol for the sensitive and specific detection of all three Borrelia burgdorferi genospecies pathogenic to humans in Lyme borreliosis patients. A rapid DNA extraction method using alkaline lysis was introduced and was found to be superior to other DNA extraction methods. Nested PCR was performed with primer sets targeting the plasmid-located ospA gene and a chromosomal gene segment encoding a 66-kDa protein (p66). In spiked synovial fluid (SF) fewer than three borreliae/sample were detected. The specificities of the amplicons were confirmed by Southern blot analysis with PCR-derived probes. Urine, cerebrospinal fluid (CSF), and SF specimens from 57 patients with Lyme borreliosis and from 58 controls were examined. In clinical samples the diagnostic sensitivity of PCR was 85% with SF samples, 79% with urine samples, and 91% with paired SF-urine samples from patients with Lyme arthritis and was 79% with CSF samples, 45% with urine samples, and 87% with paired CSF-urine specimens from neuroborreliosis patients. One patient each with neuroborreliosis and with Lyme arthritis had PCRpositive urine samples only. In 17% of all cases both primer sets yielded positive results, while the other patients were positive with only one primer set. Among these, more positive results were obtained with the p66 gene primer than with the ospA primer. The specificity exceeded 99%. We conclude that DNA from B. burgdorferi sensu lato species can sensitively and specifically be detected with the optimized PCR method described. At least two different primer sets should be used, and whenever possible, urine and CSF or SF should be analyzed in parallel to achieve maximum sensitivity of the test. This protocol, therefore, considerably enhances the diagnostic power of PCR in patients with B. burgdorferi infection.

Evaluation of an internally controlled real-time PCR targeting the ospA gene for detection of Borrelia burgdorferi sensu lato DNA in cerebrospinal fluid

“…Borrelia DNA was isolated efficiently using QIAamp nucleic acid extraction columns [26], and the sensitivity of the assay with clinical specimens obtained from ten neuroborreliosis patients (group II) was 50%. This sensitivity was similar to that of previously described nested PCR (47%) and standard PCR (38%) assays targeting ospA [19,25], thereby providing the first description of a sensitive clinically relevant real-time PCR for detection of B. burgdorferi sensu lato in CSF samples.…”

“…Numerous PCR assays have been described previously for the detection of B. burgdorferi sensu lato DNA in CSF, but the reported sensitivities have varied from 12% to 100% [8][9][10][11][12][13][14][16][17][18][19][20][21][22][23][24][25][26][27]. These results are difficult to interpret because of the use of small sample sizes, the differences in selection of clinical specimens, the testing of poorly defined patient categories, the occasional use of insensitive assays, and the frequent lack of an internal control to monitor PCR inhibition.…”

“…Different genes have been selected for use as a target for detection, including the p66, 16S rRNA, flagellin and 2H1 genes, the 30-kb circular plasmid, and the plasmid-encoded ospA ⁄ B gene [8][9][10][11][12][13][14][16][17][18][19][20][21][22][23][24][25][26][27]. The highest sensitivities with CSF have been found in studies using the ospA ⁄ B plasmid gene [14,18,19,23,24]. The ospA gene is a fully sequenced plasmid gene specific for B. burgdorferi sensu lato species [1], including B. valaisiana [28].…”

Global Tn‐seq analysis of carbohydrate utilization and vertebrate infectivity of Borrelia burgdorferi

“…were spun down and genomic DNA was isolated from the cells using alkaline lysis as described previously (Priem et al, 1997). Briefly, the cultures were spun down at 12,000 rcf for 5 min and resuspended in 50 ml 50 mM NaOH.…”

“…The mixture was added to BSK-Lite or BSK-Lite supplemented with the carbohydrate being tested in a 1.5 ml reaction tube to a final concentration of 1 3 10 5 bacteria/ml. To determine the ratio of mutant bacteria to wild-type in the initial inoculum, 10 7 bacteria were spun down and the pellet frozen at 280 C. At five days postinoculation, genomic DNA was isolated from the mixed cultures as well as the pellet of the starting mixture using alkaline lysis as described above (Priem et al, 1997). DNA was stored at 280 C.…”