Author/Editor		Picken, MM; Picken, RN; Han, D; Cheng, Y; Strle, F
Title		Single-tube nested polymerase chain reaction assay based on flagellin gene sequences for detection of Borrelia burgdorferi sensu lato
Type		članek
Source		Eur J Clin Microbiol Infect Dis
Vol. and No.		Letnik 15, št. 6
Publication year		1996
Volume		str. 489-98
Language		eng
Abstract		An inherent drawback of nested PCR systems to increase sensitivity of PCR-based assays is that tubes must be opened after the first round of amplification in order to transfer template molecules to the second amplification reaction; this procedure introduces the risk of carry-over contamination of negative specimens. To obviate this disadvantage, a nested PCR assay for detection of Borrelia burgdorferi in which both amplifications are performed in a single tube that remains closed throughout the entire process was devised. The assay is based on flagellin gene sequences with previously determined species-wide and species-specific properties. The nested PCR system proved to be 1000 times more sensitive tha the conventional assay. Using the nested PCR system, ten spirochaetes could be routinely detected by agarose gel electrophoresis alone, whereas the conventional PCR system could detect only 10/4 spirochaetes under these conditions. After Southern transfer of amplification products and hybridization with 32P- or chemiluminescent-labeled probes, the nested PCR system could easily detect a single spirochaete by both means, whereas the sensitivity of the conventional PCR assay varied from 10/1 (32P) to 10/3 (chemiluminescence) spirochaetes. This single-tube nested PCR system should be a useful addition to the current range of diagnostic assays for Lyme borreliosis.
Descriptors		BORRELIA BURGDORFERI FLAGELLIN POLYMERASE CHAIN REACTION MAGNESIUM MOLECULAR SEQUENCE DATA SENSITIVITY AND SPECIFICITY BASE SEQUENCE