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Single-tube nested polymerase chain reaction assay based on flagellin gene sequences for detection ofBorrelia burgdorferi sensu lato

  • M. M. Picken
  • R. N. Picken
  • D. Han
  • Y. Cheng
  • F. Strle
Articles

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 ofBorrelia 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 than 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 104 spirochaetes under these conditions. After Southern transfer of amplification products and hybridization with32P- 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 101 (32P) to 103 (chemiluminescence) spirochaetes. This single-tube nested PCR system should be a useful addition to the current range of diagnostic assays for Lyme borreliosis.

Keywords

Polymerase Chain Reaction Assay Nest Polymerase Chain Reaction Template Molecule Lyme Borreliosis Diagnostic Assay 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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Copyright information

© MMV Medizin Verlag GmbH 1996

Authors and Affiliations

  • M. M. Picken
    • 1
    • 2
  • R. N. Picken
    • 3
  • D. Han
    • 3
  • Y. Cheng
    • 3
  • F. Strle
    • 4
  1. 1.Department of Pathology, Room 2242 Building 110Loyola University Medical CenterMaywoodUSA
  2. 2.Hines Veterans' Administration HospitalMaywoodUSA
  3. 3.Section of Infectious DiseaseRush-Presbyterian-St. Luke's Medical CenterChicagoUSA
  4. 4.Department of Infectious DiseasesUniversity Medical CentreLjubljanaSlovenia

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