RT Journal Article
T1 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.
A1 Queipo-Ortuño, Maria Isabel
A1 Colmenero, Juan de Dios
A1 Macias, Manuel
A1 Bravo, Maria Jose
A1 Morata, Pilar
K1 Brucelosis
K1 ADN bacteriano
K1 Inmunotransferencia Western
K1 Electroforesis en gel de poliacrilamida
K1 Inhibidores enzimáticos
K1 Inmunoglobulina G
K1 Reacción en cadena de la polimerasa
K1 Suero
AB 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.
PB American Society for Microbiology
SN 1556-6811
YR 2008
FD 2008-02
LK http://hdl.handle.net/10668/1578
UL http://hdl.handle.net/10668/1578
LA en
NO 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
NO Journal Article; Research Support, Non-U.S. Gov't;
DS RISalud
RD Apr 11, 2025