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Differential susceptibility of PCR reactions to inhibitors: an important and unrecognised phenomenon

Jim F Huggett1*, Tanya Novak1, Jeremy A Garson2, Clare Green1, Stephen D Morris-Jones3, Robert F Miller4 and Alimuddin Zumla1

Author Affiliations

1 Centre for Infectious Diseases and International Health, Windeyer Institute for Medical Sciences, 46 Cleveland Street, University College London, London, W1T 4JF, UK

2 Centre for Virology, Windeyer Institute for Medical Sciences, University College London, London, W1T 4JF, UK

3 Department of Microbiology, University College London Hospitals NHS Foundation Trust, Windeyer Institute for Medical Sciences, London, W1T 4JF, UK

4 Research Department of Infection and Population Health, Division of Population Health, University College London, London, WC1E 6JB, UK

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BMC Research Notes 2008, 1:70  doi:10.1186/1756-0500-1-70

Published: 28 August 2008

Abstract

Background

PCR inhibition by nucleic acid extracts is a well known yet poorly described phenomenon. Inhibition assessment generally depends on the assumption that inhibitors affect all PCR reactions to the same extent; i.e. that the reaction of interest and the control reaction are equally susceptible to inhibition. To test this assumption we performed inhibition assessment on DNA extracts from human urine samples, fresh urine and EDTA using different PCR reactions.

Results

When copurified inhibitors were assessed using two different PCR reactions one reaction appeared to be inhibited whilst the other was not. Further experiments using various concentrations of unextracted urine to inhibit six different PCR reactions revealed that susceptibility to inhibition was highly variable between reactions. Similar results were obtained using EDTA as the PCR inhibitor. We could find no obvious explanation why one reaction should be more susceptible to inhibition than another, although a possible association with amplicon GC content was noted.

Conclusion

These findings have serious implications for all PCR-based gene expression studies, including the relatively new PCR array method, and for both qualitative and quantitative PCR-based molecular diagnostic assays, suggesting that careful consideration should be given to inhibition compatibility when conducting PCR analyses. We have demonstrated unequivocally that it is not safe to assume that different PCR reactions are equally susceptible to inhibition by substances co-purified in nucleic acid extracts.