title: Preparation of bacterial DNA template by boiling and effect of immunoglobulin G as an inhibitor in real-time PCR for serum samples from patients with brucellosis.
abstract: Real-time PCR is a widely used tool for the diagnosis of many infectious diseases. However, little information exists about the influences of the different factors involved in PCR on the amplification efficiency. The aim of this study was to analyze the effect of boiling as the DNA preparation method on the efficiency of the amplification process of real-time PCR for the diagnosis of human brucellosis with serum samples. Serum samples from 10 brucellosis patients were analyzed by a SYBR green I LightCycler-based real-time PCR and by using boiling to obtain the DNA. DNA prepared by boiling lysis of the bacteria isolated from serum did not prevent the presence of inhibitors, such as immunoglobulin G (IgG), which were extracted with the template DNA. To identify and confirm the presence of IgG, serum was precipitated to separate and concentrate the IgG and was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. The use of serum volumes above 0.6 ml completely inhibited the amplification process. The inhibitory effect of IgG in serum samples was not concentration dependent, and it could be eliminated by diluting the samples 1/10 and 1/20 in water. Despite the lack of the complete elimination of the IgG from the template DNA, boiling does not require any special equipment and it provides a rapid, reproducible, and cost-effective method for the preparation of DNA from serum samples for the diagnosis of brucellosis.
authors: Biochemistry and Molecular Biology Department, Faculty of Medicine, Malaga, Spain. iqueipo@uma.es
authors: Maria Isabel
authors: MI
authors: Queipo-Ortuño
authors: Juan
authors: J
authors: De Dios Colmenero
authors: Manuel
authors: M
authors: Macias
authors: Maria Jose
authors: MJ
authors: Bravo
authors: Pilar
authors: P
authors: Morata
mesh_terms: D015153:Blotting, Western; D002006:Brucellosis / Q000175:diagnosis*; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D007074:Immunoglobulin G / Q000494:pharmacology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D044967:Serum / Q000737:chemistry / Q000382:microbiology*
chemical_list: D004269:DNA, Bacterial; D004791:Enzyme Inhibitors; D007074:Immunoglobulin G