In this study, the Agrobacterium tumefaciens-mediated transformation method for Ganderma weberianum has been established. Driven by the cauliflower mosaic virus (CaMV) 35S promoter, the hygromycin phosphotransferase (hpt), β-glucuronidase (uidA), and enhanced green fluorescent protein (egfp) genes have been efficiently expressed in transgenic mycelia and spores. The transformation system was composed of the growing mycelia, A. tumefaciens strain GV3101, and the expression vector pBI-H1, harboring the CaMV 35S promoter and selective hpt marker. The genetic transformation of G. weberianum was achieved through co-cultivation of Agrobacterium lawn and fungal mycelia at 28 °C on yeast extract agar (YEA) medium. Stable genetic transformants were obtained through successive hygromycin B selections and single spore isolation. Over 80 % of transformants showed genetic stability even after ten rounds of subculturing. The simple and efficient genetic transformation method is a useful tool for molecular genetics analyses and gene manipulation of G. weberianum.

• MeSH
• Agrobacterium tumefaciens genetics   metabolism   MeSH
• Ganoderma genetics   MeSH
• Genetic Techniques * MeSH
• Genetic Vectors genetics   metabolism   MeSH
• Transformation, Genetic * MeSH
• Green Fluorescent Proteins MeSH
• Publication type
• Journal Article MeSH
• Evaluation Study MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• enhanced green fluorescent protein MeSH Browser
• Green Fluorescent Proteins MeSH

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.

• MeSH
• Antigens, Surface genetics   MeSH
• Bacterial Vaccines MeSH
• Benzothiazoles MeSH
• Borrelia burgdorferi Group classification   genetics   immunology   isolation & purification   MeSH
• Quinolines MeSH
• Diamines MeSH
• DNA, Bacterial analysis   isolation & purification   MeSH
• Electrophoresis, Agar Gel MeSH
• Enzyme-Linked Immunosorbent Assay MeSH
• Genotype MeSH
• Immunoglobulin G blood   MeSH
• Immunoglobulin M blood   MeSH
• Humans MeSH
• Lipoproteins genetics   MeSH
• Lyme Disease diagnosis   MeSH
• Cerebrospinal Fluid microbiology   MeSH
• Organic Chemicals metabolism   MeSH
• Polymerase Chain Reaction methods   MeSH
• Bacterial Outer Membrane Proteins genetics   MeSH
• Antibodies, Bacterial blood   MeSH
• Sensitivity and Specificity MeSH
• Serum microbiology   MeSH
• Bacterial Typing Techniques MeSH
• Blotting, Western MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Antigens, Surface MeSH
• Bacterial Vaccines MeSH
• Benzothiazoles MeSH
• Quinolines MeSH
• Diamines MeSH
• DNA, Bacterial MeSH
• Immunoglobulin G MeSH
• Immunoglobulin M MeSH
• Lipoproteins MeSH
• Organic Chemicals MeSH
• OspA protein MeSH Browser
• Bacterial Outer Membrane Proteins MeSH
• Antibodies, Bacterial MeSH
• SYBR Green I MeSH Browser