Identification and characterization of immunoglobulin G in blood as a major inhibitor of diagnostic PCR

A major inhibitor of diagnostic PCR in human plasma was identified and the mechanism of inhibition was characterized. Human blood was divided by centr ifugation into buffy coat, plasma, platelets, and erythrocytes. All these b lood fractions were found to be highly inhibitory to a standardized PCR mix ture containing the thermostable DNA polymerase AmpliTaq Gold, PCR inhibito rs in human plasma were purified by chromatographic procedures and were cha racterized by a process of elimination, so that the PCR-inhibitory effects of plasma fractions were tested after each purification step. The major inh ibitor in human plasma, as determined by size-exclusion chromatography, ani on-exchange chromatography, and chromatofocusing, was found to be immunoglo bulin G (IgG) on the basis of N-terminal amino acid sequencing and electrop horetic analysis of the purified polypeptide. When different concentrations of purified plasma IgG (PIgG) were added to PCR mixtures containing II dif ferent thermostable DNA polymerases and 1 ng of Listeria monocytogenes DNA as template DNA, the only polymerase that resisted inhibition was rTth, The inhibitory effect was reduced when PIgG was heated at 95 degrees C before it was added to PCR or after the addition of excess nontarget DNA to the PC R mixture. However, heating of PIgG together with target DNA at 95 degrees C was found to block the amplification. Inhibition by PIgG mag be due to an interaction with single-stranded. DNA, which makes the target DNA unavaila ble for 10 of the DNA polymerases tested, The results show the danger of us ing boiling as a method of sample pretreatment or using a hot start prior t o PCR. The effect of plasma PCR inhibition could be removed by mixing plasm a with DNA-agarose beads prior to amplification, while plasma PCR inhibitor s were found to bind to the DNA-agarose beads.