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.


Autoria(s): Queipo-Ortuño, Maria Isabel; Colmenero, Juan de Dios; Macias, Manuel; Bravo, Maria Jose; Morata, Pilar
Data(s)

11/04/2014

11/04/2014

01/02/2008

Resumo

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.

Journal Article; Research Support, Non-U.S. Gov't;

This study was supported by Red Tematica para la Investigacion en Brucelosi, Instituto de Salud Carlos III (ISCIII), grant PI 05/1733; Servicio Andaluz de Salud (SAS) grant 152/04; and Consejeria de Innovación Ciencia y Empresa (GI 2005, CTS-276).

Identificador

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

1556-679X (Online)

1556-6811 (Print)

PMC2238042

http://hdl.handle.net/10668/1578

18077622

10.1128/CVI.00270-07

Idioma(s)

en

Publicador

American Society for Microbiology

Relação

Clinical and vaccine immunology : CVI

http://cvi.asm.org/content/15/2/293.abstract

Direitos

Acceso abierto

Palavras-Chave #Brucelosis #ADN bacteriano #Inmunotransferencia Western #Electroforesis en gel de poliacrilamida #Inhibidores enzimáticos #Inmunoglobulina G #Reacción en cadena de la polimerasa #Suero #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
Tipo

info:eu-repo/semantics/article

info:eu-repo/semantics/published

Artículo