Inhibition of polymerase chain reaction: Pathogen-specific controls are better than human gene amplification

Autoři: Guillaume Roux aff001;  Christophe Ravel aff002;  Emmanuelle Varlet-Marie aff002;  Rachel Jendrowiak aff002;  Patrick Bastien aff002;  Yvon Sterkers aff002
Působiště autorů: Univ. Montpellier, and Laboratoire de Microbiologie, Centre Hospitalier Universitaire (CHU) of Nîmes, Nîmes, France aff001;  Univ. Montpellier, Centre Hospitalier Universitaire (CHU) of Montpellier, Dept. of Parasitology-Mycology, Research Unit MiVEGEC, CNRS, IRD, Montpellier, France aff002
Vyšlo v časopise: PLoS ONE 14(9)
Kategorie: Research Article
doi: 10.1371/journal.pone.0219276


PCR inhibition is frequent in medical microbiology routine practice and may lead to false-negative results; however there is no consensus on how to detect it. Pathogen-specific and human gene amplifications are widely used to detect PCR inhibition. We aimed at comparing the value of PCR inhibitor detection using these two methods. We analysed Cp shifts (ΔCp) obtained from qPCRs targeting either the albumin gene or the pathogen-specific sequence used in two laboratory-developed microbiological qPCR assays. 3152 samples including various matrixes were included. Pathogen-specific amplification and albumin qPCR identified 62/3152 samples (2.0%), and 409/3152 (13.0%) samples, respectively, as inhibited. Only 16 samples were detected using both methods. In addition, the use of the Youden's index failed to determine adequate Cp thresholds for albumin qPCR, even when we distinguished among the different sample matrixes. qPCR targeting the albumin gene therefore appears not adequate to identify the presence of PCR inhibitors in microbiological PCR assays. Our data may be extrapolated to other heterologous targets and should discourage their use to assess the presence of PCR inhibition in microbiological PCR assays.

Klíčová slova:

Albumins – Cerebrospinal fluid – DNA extraction – Polymerase chain reaction – Toxoplasma – White blood cells – Pneumocystis – Amniotic fluid


1. Hedman J, Radstrom P. Overcoming inhibition in real-time diagnostic PCR. Methods Mol Biol. 2013;943:17–48. Epub 2012/10/30. doi: 10.1007/978-1-60327-353-4_2 23104280.

2. Wallace PS, MacKay WG. Quality in the molecular microbiology laboratory. Methods Mol Biol. 2013;943:49–79. Epub 2012/10/30. doi: 10.1007/978-1-60327-353-4_3 23104281.

3. Schrader C, Schielke A, Ellerbroek L, Johne R. PCR inhibitors—occurrence, properties and removal. J Appl Microbiol. 2012;113(5):1014–26. Epub 2012/07/04. doi: 10.1111/j.1365-2672.2012.05384.x 22747964.

4. Buckwalter SP, Sloan LM, Cunningham SA, Espy MJ, Uhl JR, Jones MF, et al. Inhibition controls for qualitative real-time PCR assays: are they necessary for all specimen matrices? J Clin Microbiol. 2014;52(6):2139–43. Epub 2014/04/18. doi: 10.1128/JCM.03389-13 24740078; PubMed Central PMCID: PMC4042775.

5. Doskaya M, Caner A, Degirmenci A, Wengenack NL, Yolasigmaz A, Turgay N, et al. Degree and frequency of inhibition in a routine real-time PCR detecting Pneumocystis jirovecii for the diagnosis of Pneumocystis pneumonia in Turkey. J Med Microbiol. 2011;60(Pt 7):937–44. Epub 2011/04/05. doi: 10.1099/jmm.0.030775-0 21459903.

6. Bustin SA, Beaulieu JF, Huggett J, Jaggi R, Kibenge FS, Olsvik PA, et al. MIQE precis: Practical implementation of minimum standard guidelines for fluorescence-based quantitative real-time PCR experiments. BMC Mol Biol. 11. England2010. p. 74.

7. Sterkers Y, Varlet-Marie E, Marty P, Bastien P. Diversity and evolution of methods and practices for the molecular diagnosis of congenital toxoplasmosis in France: a 4-year survey. Clin Microbiol Infect. 2010;16(10):1594–602. doi: 10.1111/j.1469-0691.2009.03101.x 19886905.

8. Rosenstraus M, Wang Z, Chang SY, DeBonville D, Spadoro JP. An internal control for routine diagnostic PCR: design, properties, and effect on clinical performance. J Clin Microbiol. 1998;36(1):191–7. Epub 1998/02/12. 9431945; PubMed Central PMCID: PMC124832.

9. Maaroufi Y, de Bruyne JM, Duchateau V, Scheen R, Crokaert F. Development of a multiple internal control for clinical diagnostic real-time amplification assays. FEMS Immunol Med Microbiol. 2006;48(2):183–91. Epub 2006/10/27. doi: 10.1111/j.1574-695X.2006.00125.x 17064275.

10. Brenier-Pinchart MP, Morand-Bui V, Fricker-Hidalgo H, Equy V, Marlu R, Pelloux H. Adapting a conventional PCR assay for Toxoplasma gondii detection to real-time quantitative PCR including a competitive internal control. Parasite. 2007;14(2):149–54. Epub 2007/07/25. doi: 10.1051/parasite/2007142149 17645187.

11. Daval S, Poirier P, Armenaud J, Cambon M, Livrelli V. [Development of a real-time PCR assay for quantitative diagnosis of Toxoplasma gondii after allogeneic bone marrow transplantation]. Pathol Biol (Paris). 2010;58(1):104–9. Epub 2009/11/07. doi: 10.1016/j.patbio.2009.07.013 19892493

12. Huggett JF, Novak T, Garson JA, Green C, Morris-Jones SD, Miller RF, et al. Differential susceptibility of PCR reactions to inhibitors: an important and unrecognised phenomenon. BMC Res Notes. 2008;1:70. Epub 2008/08/30. doi: 10.1186/1756-0500-1-70 18755023; PubMed Central PMCID: PMC2564953.

13. Pionzio AM, McCord BR. The effect of internal control sequence and length on the response to PCR inhibition in real-time PCR quantitation. Forensic Sci Int Genet. 2014;9:55–60. Epub 2014/02/18. doi: 10.1016/j.fsigen.2013.10.010 24528580.

14. Chabbert E, Lachaud L, Crobu L, Bastien P. Comparison of two widely used PCR primer systems for detection of toxoplasma in amniotic fluid, blood, and tissues. J Clin Microbiol. 2004;42(4):1719–22. 173. doi: 10.1128/JCM.42.4.1719-1722.2004 15071031

15. Hohlfeld P, Daffos F, Costa JM, Thulliez P, Forestier F, Vidaud M. Prenatal diagnosis of congenital toxoplasmosis with a polymerase-chain-reaction test on amniotic fluid. N Engl J Med. 1994;331(11):695–9. Epub 1994/09/15. doi: 10.1056/NEJM199409153311102 8058075.

16. Sterkers Y, Pratlong F, Albaba S, Loubersac J, Picot MC, Pretet V, et al. Novel interpretation of molecular diagnosis of congenital toxoplasmosis according to gestational age at the time of maternal infection. J Clin Microbiol. 2012;50(12):3944–51. Epub 2012/10/05. doi: 10.1128/JCM.00918-12 23035201; PubMed Central PMCID: PMC3502944.

17. Delhaes L, Filisetti D, Brenier-Pinchart MP, Pelloux H, Yera H, Dalle F, et al. Freezing and storage at -20 degrees C provides adequate preservation of Toxoplasma gondii DNA for retrospective molecular analysis. Diagn Microbiol Infect Dis. 2014;80(3):197–9. Epub 2014/09/23. doi: 10.1016/j.diagmicrobio.2014.08.007 25241642.

18. Reischl U, Bretagne S, Kruger D, Ernault P, Costa JM. Comparison of two DNA targets for the diagnosis of Toxoplasmosis by real-time PCR using fluorescence resonance energy transfer hybridization probes. BMC Infect Dis. 2003;3:7. Epub 2003/05/06. doi: 10.1186/1471-2334-3-7 12729464; PubMed Central PMCID: PMC156600.

19. Fillaux J, Berry A. Real-time PCR assay for the diagnosis of Pneumocystis jirovecii pneumonia. Methods Mol Biol. 2013;943:159–70. Epub 2012/10/30. doi: 10.1007/978-1-60327-353-4_11 23104289.

20. Westgard JO, Barry PL, Hunt MR, Groth T. A multi-rule Shewhart chart for quality control in clinical chemistry. Clin Chem. 1981;27(3):493–501. Epub 1981/03/01. 7471403.

21. Mary C, Faraut F, Lascombe L, Dumon H. Quantification of Leishmania infantum DNA by a real-time PCR assay with high sensitivity. J Clin Microbiol. 2004;42(11):5249–55. Epub 2004/11/06. doi: 10.1128/JCM.42.11.5249-5255.2004 15528722; PubMed Central PMCID: PMC525214.

22. Varlet-Marie E, Sterkers Y, Brenier-Pinchart MP, Cassaing S, Dalle F, Delhaes L, et al. Characterization and multicentric validation of a common standard for Toxoplasma gondii detection using nucleic acid amplification assays. J Clin Microbiol. 2014;52(11):3952–9. Epub 2014/09/05. doi: 10.1128/JCM.01906-14 25187637; PubMed Central PMCID: PMC4313224.

23. Kralik P, Ricchi M. A Basic Guide to Real Time PCR in Microbial Diagnostics: Definitions, Parameters, and Everything. Front Microbiol. 2017;8:108. Epub 2017/02/18. doi: 10.3389/fmicb.2017.00108 28210243; PubMed Central PMCID: PMC5288344.

24. RCore-Team. R: a language and environment for statistical computing: R Foundation for Statistical Computing, Vienna, Austria.; 2017. Available from:

25. RStudio-Team. R Studio: Integrated Development for R.: RStudio, Inc., Boston, MA; 2015. Available from:

26. Al-Soud WA, Radstrom P. Purification and characterization of PCR-inhibitory components in blood cells. J Clin Microbiol. 2001;39(2):485–93. Epub 2001/02/07. doi: 10.1128/JCM.39.2.485-493.2001 11158094; PubMed Central PMCID: PMC87763.

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