Publication: 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.
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Identifiers
Date
2008-02
Authors
Queipo-Ortuño, Maria Isabel
Colmenero, Juan de Dios
Macias, Manuel
Bravo, Maria Jose
Morata, Pilar
Advisors
Journal Title
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Publisher
American Society for Microbiology
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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.
Description
Journal Article; Research Support, Non-U.S. Gov't;
MeSH Terms
Medical Subject Headings::Diseases::Bacterial Infections and Mycoses::Bacterial Infections::Gram-Negative Bacterial Infections::Brucellosis
Medical Subject Headings::Chemicals and Drugs::Nucleic Acids, Nucleotides, and Nucleosides::Nucleic Acids::DNA::DNA, Bacterial
Medical Subject Headings::Analytical, Diagnostic and Therapeutic Techniques and Equipment::Investigative Techniques::Electrochemical Techniques::Electrophoresis::Electrophoresis, Polyacrylamide Gel
Medical Subject Headings::Chemicals and Drugs::Chemical Actions and Uses::Pharmacologic Actions::Molecular Mechanisms of Pharmacological Action::Enzyme Inhibitors
Medical Subject Headings::Organisms::Eukaryota::Animals::Chordata::Vertebrates::Mammals::Primates::Haplorhini::Catarrhini::Hominidae::Humans
Medical Subject Headings::Chemicals and Drugs::Amino Acids, Peptides, and Proteins::Proteins::Blood Proteins::Immunoproteins::Immunoglobulins::Antibodies::Immunoglobulin Isotypes::Immunoglobulin G
Medical Subject Headings::Analytical, Diagnostic and Therapeutic Techniques and Equipment::Investigative Techniques::Genetic Techniques::Nucleic Acid Amplification Techniques::Polymerase Chain Reaction
Medical Subject Headings::Anatomy::Hemic and Immune Systems::Blood::Serum
Medical Subject Headings::Analytical, Diagnostic and Therapeutic Techniques and Equipment::Investigative Techniques::Immunologic Techniques::Immunoassay::Immunoblotting::Blotting, Western
Medical Subject Headings::Chemicals and Drugs::Nucleic Acids, Nucleotides, and Nucleosides::Nucleic Acids::DNA::DNA, Bacterial
Medical Subject Headings::Analytical, Diagnostic and Therapeutic Techniques and Equipment::Investigative Techniques::Electrochemical Techniques::Electrophoresis::Electrophoresis, Polyacrylamide Gel
Medical Subject Headings::Chemicals and Drugs::Chemical Actions and Uses::Pharmacologic Actions::Molecular Mechanisms of Pharmacological Action::Enzyme Inhibitors
Medical Subject Headings::Organisms::Eukaryota::Animals::Chordata::Vertebrates::Mammals::Primates::Haplorhini::Catarrhini::Hominidae::Humans
Medical Subject Headings::Chemicals and Drugs::Amino Acids, Peptides, and Proteins::Proteins::Blood Proteins::Immunoproteins::Immunoglobulins::Antibodies::Immunoglobulin Isotypes::Immunoglobulin G
Medical Subject Headings::Analytical, Diagnostic and Therapeutic Techniques and Equipment::Investigative Techniques::Genetic Techniques::Nucleic Acid Amplification Techniques::Polymerase Chain Reaction
Medical Subject Headings::Anatomy::Hemic and Immune Systems::Blood::Serum
Medical Subject Headings::Analytical, Diagnostic and Therapeutic Techniques and Equipment::Investigative Techniques::Immunologic Techniques::Immunoassay::Immunoblotting::Blotting, Western
DeCS Terms
CIE Terms
Keywords
Brucelosis, ADN bacteriano, Inmunotransferencia Western, Electroforesis en gel de poliacrilamida, Inhibidores enzimáticos, Inmunoglobulina G, Reacción en cadena de la polimerasa, Suero
Citation
Queipo-Ortuño MI, De Dios Colmenero J, Macias M, Bravo MJ, Morata P. 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. Clin. Vaccine Immunol. 2008; 15(2):293-6