Improved method of detection and molecular typing of Borrelia burgdorferi sensu lato in clinical samples by polymerase chain reaction without DNA purification.
A simple assay by polymerase chain reaction was used for the of detection of Borrelia burgdorferi, causative agent of Lyme borreliosis (LB). It involves no DNA purification and is based on the amplification of a specific region of ospA gene of B. 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 which B. 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 Ceské Budejovice (South Bohemia, Czechia) showed infection with B. burgdorferi sensu stricto in 11% and B. garinii in 15% of cases. The highest prevalence was found for B. afzelii (43%). Co-infection was confirmed in 24 % of the analyzed symplex; 7% of samples that were B. burgdorferi sensu lato positive gave no results in DNA amplification with B. burgdorferi sensu stricto-, B. garinii- and B. 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.
Rudenko
N
N
Faculty of Biological Sciences, University of South Bohemia, 370 05 Ceské Budejovice, Czechia. natasha@paru.cas.cz
Golovchenko
M
M
Nemec
J
J
Volkaert
J
J
Mallátová
N
N
Grubhoffer
L
L
D000907:Antibodies, Bacterial / Q000097:blood; D000954:Antigens, Surface / Q000235:genetics; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics; D015373:Bacterial Typing Techniques; D001428:Bacterial Vaccines; D052160:Benzothiazoles; D015153:Blotting, Western; D015748:Borrelia burgdorferi Group / Q000145:classification* / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D002555:Cerebrospinal Fluid / Q000382:microbiology; D004269:DNA, Bacterial / Q000032:analysis* / Q000302:isolation & purification; D003959:Diamines; D004587:Electrophoresis, Agar Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005838:Genotype; D006801:Humans; D007074:Immunoglobulin G / Q000097:blood; D007075:Immunoglobulin M / Q000097:blood; D008074:Lipoproteins / Q000235:genetics; D008193:Lyme Disease / Q000175:diagnosis*; D009930:Organic Chemicals / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods*; D011804:Quinolines; D012680:Sensitivity and Specificity; D044967:Serum / Q000382:microbiology
D000907:Antibodies, Bacterial; D000954:Antigens, Surface; D001425:Bacterial Outer Membrane Proteins; D001428:Bacterial Vaccines; D052160:Benzothiazoles; D004269:DNA, Bacterial; D003959:Diamines; D007074:Immunoglobulin G; D007075:Immunoglobulin M; D008074:Lipoproteins; D009930:Organic Chemicals; C072045:OspA protein; D011804:Quinolines; C098022:SYBR Green I