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Journal of Clinical Microbiology, January 2000, p. 345-350, Vol. 38, No. 1
0095-1137/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Identification and Characterization of Immunoglobulin G in Blood as a Major Inhibitor of Diagnostic PCR

Waleed Abu Al-Soud, Leif J. Jönsson, and Peter Rådström*

Applied Microbiology, Center for Chemistry and Chemical Engineering, Lund Institute of Technology, Lund University, SE-221 00 Lund, Sweden

Received 17 June 1999/Returned for modification 21 July 1999/Accepted 16 September 1999

A major inhibitor of diagnostic PCR in human plasma was identified and the mechanism of inhibition was characterized. Human blood was divided by centrifugation into buffy coat, plasma, platelets, and erythrocytes. All these blood fractions were found to be highly inhibitory to a standardized PCR mixture containing the thermostable DNA polymerase AmpliTaq Gold. PCR inhibitors in human plasma were purified by chromatographic procedures and were characterized by a process of elimination, so that the PCR-inhibitory effects of plasma fractions were tested after each purification step. The major inhibitor in human plasma, as determined by size-exclusion chromatography, anion-exchange chromatography, and chromatofocusing, was found to be immunoglobulin G (IgG) on the basis of N-terminal amino acid sequencing and electrophoretic analysis of the purified polypeptide. When different concentrations of purified plasma IgG (PIgG) were added to PCR mixtures containing 11 different 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°C before it was added to PCR or after the addition of excess nontarget DNA to the PCR mixture. However, heating of PIgG together with target DNA at 95°C was found to block the amplification. Inhibition by PIgG may be due to an interaction with single-stranded DNA, which makes the target DNA unavailable for 10 of the DNA polymerases tested. The results show the danger of using boiling as a method of sample pretreatment or using a hot start prior to PCR. The effect of plasma PCR inhibition could be removed by mixing plasma with DNA-agarose beads prior to amplification, while plasma PCR inhibitors were found to bind to the DNA-agarose beads.

* Corresponding author. Mailing address: Applied Microbiology, Center for Chemistry and Chemical Engineering, Lund Institute of Technology, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden. Phone: (46)46 222 34 12. Fax: (46)46 222 42 03. E-mail: Peter.Radstrom{at}tmb.lth.se.

Journal of Clinical Microbiology, January 2000, p. 345-350, Vol. 38, No. 1
0095-1137/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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