Preparation of bacterial DNA template by boiling and effect of immunoglobulin G as an inhibitor in real-time PCR from serum samples of patients with brucellosis

Biochemistry and Molecular Biology Department, Faculty of Medicine, Malaga, Spain; Infectious Diseases Service, Carlos Haya University Hospital, Malaga, Spain; Ciber Fisiopatología Obesidad y Nutrición (CB06/03), Instituto de Salud Carlos III, Madrid, Spain; Immunology Service, Carlos Haya University Hospital, Malaga, Spain

^* To whom correspondence should be addressed. Email: iqueipo{at}uma.es.

	Abstract

Real-time PCR is a widely used diagnostic tool for the diagnosis of many infectious diseases. However, little information exists about the influence on amplification efficiency of the different factors involved. 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 in serum samples. Serum samples from ten brucellosis patients were analyzed by SYBR Green I LightCycler-based real-time PCR using boiling to obtain the DNA. DNA prepared by boiling lysis of 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 IgG and analyzed by SDS-PAGE and western blotting. Serum volumes above 0.6 ml completely inhibited the amplification process. The inhibitory effect of serum samples by IgG was not concentration-dependent and it could be eliminated by diluting the samples 1/10 and 1/20 in water. Despite not completely eliminating IgG from template DNA, boiling does not require any special equipment and it provides a rapid, reproducible and cost-effective DNA preparation method for serum samples in the diagnosis of brucellosis.