Improved method of detection and molecular typing ofBorrelia burgdorferi sensu lato in clinical samples by polymerase chain reaction without DNA purification

摘要

A simple assay by polymerase chain reaction was used for the of detection ofBorrelia burgdorferi, causative agent of Lyme borreliosis (LB). It involves no DNA purification and is based on the amplification of a specific region ofospA gene ofB. burgdorferi, followed by direct detection of the PCR product with SYBR Green I by agarose gel electrophoresis. The method was used to analyze samples from patients with LB diagnosis, with presumable infection with the LB spirochete, those with unclear clinical symptoms and after the course of an antibiotic treatment. Spirochetal DNA was detected by PCR even in contaminated samples in whichB. burgdorferi was overgrown by fungi and other bacteria. Spirochetal DNA was detected and borrelia species was identified in cerebrospinal fluid of two patients hospitalized with the diagnosis “fever of unknown origin”. Western blot and ELISA were negative in both cases. Total analysis of 94 samples from the hospital in České Budějovice (South Bohemia, Czechia) showed infection withB. burgdorferi sensu stricto in 11 % andB. garinii in 15 % of cases. The highest prevalence was found forB. afzelii (43 %). Co-infection was confirmed in 24 % of the analyzed symplex; 7 % of samples that wereB. burgdorferi sensu lato positive gave no results in DNA amplification withB. burgdorferi sensu stricto-,B. garinii- andB. afzelii-specific primers. The proposed reliable, rapid, unexpensive and specific technique could form the basis of laboratory tests for routine detection and identification of Lyme-disease spirochete in different samples.