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

被引:158
作者
Huggett J.F. [1 ]
Novak T. [1 ]
Garson J.A. [2 ]
Green C. [1 ]
Morris-Jones S.D. [3 ]
Miller R.F. [4 ]
Zumla A. [1 ]
机构
[1] Centre for Infectious Diseases and International Health, Windeyer Institute for Medical Sciences, University College London, London, W1T 4JF
[2] Centre for Virology, Windeyer Institute for Medical Sciences, University College London, London
[3] Department of Microbiology, University College London Hospitals NHS Foundation Trust, Windeyer Institute for Medical Sciences, London
[4] Research Department of Infection and Population Health, Division of Population Health, University College London, London
关键词
Polymerase Chain Reaction; Polymerase Chain Reaction Reaction; Polymerase Chain Reaction Analysis; Nucleic Acid Extract; Polymerase Chain Reaction Inhibitor;
D O I
10.1186/1756-0500-1-70
中图分类号
学科分类号
摘要
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. © 2008 Huggett et al; licensee BioMed Central Ltd.
引用
收藏
相关论文
共 25 条
  • [1] Bustin S.A., Real-time, fluorescence-based quantitative PCR: A snapshot of current procedures and preferences, Expert Rev Mol Diagn, 5, pp. 493-498, (2005)
  • [2] Bustin S.A., Nolan T., Pitfalls of quantitative real-time reverse-transcription polymerase chain reaction, J Biomol Tech, 15, pp. 155-166, (2004)
  • [3] Nolan T., Hands R.E., Bustin S.A., Quantification of mRNA using real-time RT-PCR, Nat Protoc, 1, pp. 1559-1582, (2006)
  • [4] Tichopad A., Dilger M., Schwarz G., Pfaffl M.W., Standardized determination of real-time PCR efficiency from a single reaction set-up, Nucleic Acids Res, 31, (2003)
  • [5] Ferns R.B., Garson J.A., Development and evaluation of a real-time RT-PCR assay for quantification of cell-free human immunodeficiency virus type 2 using a Brome Mosaic Virus internal control, J Virol Methods, 135, pp. 102-108, (2006)
  • [6] Kontanis E.J., Reed F.A., Evaluation of real-time PCR amplification efficiencies to detect PCR inhibitors, J Forensic Sci, 51, pp. 795-804, (2006)
  • [7] Monpoeho S., Coste-Burel M., Costa-Mattioli M., Besse B., Chomel J.J., Billaudel S., Ferre V., Application of a real-time polymerase chain reaction with internal positive control for detection and quantification of enterovirus in cerebrospinal fluid, Eur J Clin Microbiol Infect Dis, 21, pp. 532-536, (2002)
  • [8] Nolan T., Hands R.E., Ogunkolade W., Bustin S.A., SPUD: A quantitative PCR assay for the detection of inhibitors in nucleic acid preparations, Anal Biochem, 351, pp. 308-310, (2006)
  • [9] Smith R.D., Brown B., Ikonomi P., Schechter A.N., Exogenous reference RNA for normalization of real-time quantitative PCR, Biotechniques, 34, pp. 88-91, (2003)
  • [10] Bustin S.A., Benes V., Nolan T., Pfaffl M.W., Quantitative real-time RT-PCR - A perspective, J Mol Endocrinol, 34, pp. 597-601, (2005)