SpringerLink
Log in

Amplification Product Inactivation

  • Chapter
  • First Online:
Advanced Techniques in Diagnostic Microbiology

Abstract

“With great power comes great responsibility.” This popular Spider Man movie quote infers the relationship between molecular nucleic acid amplification methods and amplification product inactivation procedures. The “power” is the ability of molecular amplification methods to produce billions of copies of nucleic acid from one template strand. This technique has been modified and adapted to serve as a means to detect viral and bacterial nucleic acids in clinical settings. The use of molecular methods is constantly evolving and expanding in its utility in this arena. With these methods, it is possible to detect small amounts of herpes simplex virus (HSV) in an infant’s cerebrospinal fluid (CSF) with suspected meningitis [1]. With the detection of HSV in the CSF, antiviral therapy can be continued with confidence that the therapy is preventing brain damage that can be caused by HSV [2]. Before the advent of nucleic acid amplification techniques, there was little to no chance of recovering the virus from this sample type with traditional viral laboratory methods [3]. Empiric antiviral therapy was either continued or stopped without objective data upon which to base the decision.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution

Subscribe and save

Springer+ Basic
EUR 32.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or Ebook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Chapter
EUR 29.95
Price includes VAT (Germany)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
EUR 213.99
Price includes VAT (Germany)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Similar content being viewed by others

References

  1. Smalling TW, Sefers SE, Li H, Tang YW (2002) Molecular approaches to detecting herpes simplex virus and enteroviruses in the central nervous system. J Clin Microbiol 40(7):2317–2322

    Article  PubMed  CAS  Google Scholar 

  2. Kimberlin D (2004) Herpes simplex virus, meningitis and encephalitis in neonates. Herpes 11(Suppl 2):65A–76A

    PubMed  Google Scholar 

  3. Long SS, Pool TE, Vodzak J, Daskalaki I, Gould JM (2011) Herpes simplex virus infection in young infants during 2 decades of Empiric Acyclovir Therapy. Pediatr Infect Dis J 30(7):556–561

    Article  PubMed  Google Scholar 

  4. Sefers SE, Pei Z, Tang YW (2005) False positives and false negatives encountered in diagnostic molecular microbiology. Rev Med Microbiol 16(2):59–67

    Article  Google Scholar 

  5. Aslanzadeh J, Preventing PCR (2004) amplification carryover contamination in a clinical laboratory. Ann Clin Lab Sci 34(4):389–396

    PubMed  CAS  Google Scholar 

  6. Borst A, Box AT, Fluit AC (2004) False-positive results and contamination in nucleic acid amplification assays: suggestions for a prevent and destroy strategy. Eur J Clin Microbiol Infect Dis 23(4):289–299

    Article  PubMed  CAS  Google Scholar 

  7. Mitchell C, Kraft K, Peterson D, Frenkel L (2010) Cross-contamination during processing of dried blood spots used for rapid diagnosis of HIV-1 infection of infants is rare and avoidable. J Virol Methods 163(2):489–491

    Article  PubMed  CAS  Google Scholar 

  8. Tang JW, Lin M, Chiu L, Koay ES (2010) Viral loads of herpes simplex virus in clinical samples–a 5-year retrospective analysis. J Med Virol 82(11):1911–1916

    Article  PubMed  Google Scholar 

  9. Stals A, Werbrouck H, Baert L et al (2009) Laboratory efforts to eliminate contamination problems in the real-time RT-PCR detection of noroviruses. J Microbiol Methods 77(1):72–76

    Article  PubMed  CAS  Google Scholar 

  10. McDermott SS, McDermott PF, Skare J et al (2000) Positive CSF HSV PCR in patients with GBM: a note of caution. Neurology 54(3):746–749

    Article  PubMed  CAS  Google Scholar 

  11. Tilburg JJ, Nabuurs-Franssen MH, van Hannen EJ, Horrevorts AM, Melchers WJ, Klaassen CH (2010) Contamination of commercial PCR master mix with DNA from Coxiella burnetii. J Clin Microbiol 48(12):4634–4635

    Article  PubMed  Google Scholar 

  12. Corless CE, Guiver M, Borrow R, Edwards-Jones V, Kaczmarski EB, Fox AJ (2000) Contamination and sensitivity issues with a real-time universal 16S rRNA PCR. J Clin Microbiol 38(5):1747–1752

    PubMed  CAS  Google Scholar 

  13. Muhl H, Kochem AJ, Disque C, Sakka SG (2010) Activity and DNA contamination of commercial polymerase chain reaction reagents for the universal 16S rDNA real-time polymerase chain reaction detection of bacterial pathogens in blood. Diagn Microbiol Infect Dis 66(1):41–49

    Article  PubMed  Google Scholar 

  14. Evans GE, Murdoch DR, Anderson TP, Potter HC, George PM, Chambers ST (2003) Contamination of Qiagen DNA extraction kits with Legionella DNA. J Clin Microbiol 41(7):3452–3453

    Article  PubMed  CAS  Google Scholar 

  15. Monleau M, Plantier JC, Peeters M (2010) HIV contamination of commercial PCR enzymes raises the importance of quality control of low-cost in-house genotypic HIV drug resistance tests. Antivir Ther 15(1):121–126

    Article  PubMed  CAS  Google Scholar 

  16. Prince AM, Andrus L (1992) PCR: how to kill unwanted DNA. Biotechniques 12(3):358–360

    PubMed  CAS  Google Scholar 

  17. Khlif M, Mary C, Sellami H et al (2009) Evaluation of nested and real-time PCR assays in the diagnosis of candidaemia. Clin Microbiol Infect 15(7):656–661

    Article  PubMed  CAS  Google Scholar 

  18. Sarkar G, Sommer S (1990) More light on PCR contamination. Nature 347(6291):340–341

    Article  PubMed  CAS  Google Scholar 

  19. Kwok S, Higuchi R (1989) Avoiding false positives with PCR. Nature 339(6221):237–238

    Article  PubMed  CAS  Google Scholar 

  20. Ou CY, Moore JL, Schochetman G (1991) Use of UV irradiation to reduce false positivity in polymerase chain reaction. Biotechniques 10(4):442, 444, 446

    Google Scholar 

  21. Belak S, Ballagi-Pordany A (1993) Experiences on the application of the polymerase chain reaction in a diagnostic laboratory. Mol Cell Probes 7(3):241–248

    Article  PubMed  CAS  Google Scholar 

  22. Longo MC, Berninger MS, Hartley JL (1990) Use of uracil DNA glycosylase to control carry-over contamination in polymerase chain reactions. Gene 93(1):125–128

    Article  PubMed  CAS  Google Scholar 

  23. Rys PN, Persing DH (1993) Preventing false positives: quantitative evaluation of three protocols for inactivation of polymerase chain reaction amplification products. J Clin Microbiol 31(9):2356–2360

    PubMed  CAS  Google Scholar 

  24. Espy MJ, Smith TF, Persing DH (1993) Dependence of polymerase chain reaction product inactivation protocols on amplicon length and sequence composition. J Clin Microbiol 31(9):2361–2365

    PubMed  CAS  Google Scholar 

  25. Kleiboeker SB (2005) Quantitative assessment of the effect of uracil-DNA glycosylase on amplicon DNA degradation and RNA amplification in reverse transcription-PCR. Virol J 2:29

    Article  PubMed  Google Scholar 

  26. Taggart EW, Carroll KC, Byington CL, Crist GA, Hillyard DR (2002) Use of heat labile UNG in an RT-PCR assay for enterovirus detection. J Virol Methods 105(1):57–65

    Article  PubMed  CAS  Google Scholar 

  27. Pierce KE, Wangh LJ (2004) Effectiveness and limitations of uracil-DNA glycosylases in sensitive real-time PCR assays. Biotechniques 36(1):44–46, 48

    Google Scholar 

  28. Martins TB, Hillyard DR, Litwin CM, Taggart EW, Jaskowski TD, Hill HR (2000) Evaluation of a PCR probe capture assay for the detection of Toxoplasma gondii. Incorporation of uracil N-glycosylase for contamination control. Am J Clin Pathol 113(5):714–721

    Article  PubMed  CAS  Google Scholar 

  29. Mohamed N, Elfaitouri A, Fohlman J, Friman G, Blomberg J (2004) A sensitive and quantitative single-tube real-time reverse transcriptase-PCR for detection of enteroviral RNA. J Clin Virol 30(2):150–156

    Article  PubMed  CAS  Google Scholar 

  30. Kao SY, Niemiec TM, Loeffelholz MJ, Dale B, Rotbart HA (1995) Direct and uninterrupted RNA amplification of enteroviruses with colorimetric microwell detection. Clin Diagn Virol 3(3):247–257

    Article  PubMed  CAS  Google Scholar 

  31. Sarmiento OL, Weigle KA, Alexander J, Weber DJ, Miller WC (2003) Assessment by meta-analysis of PCR for diagnosis of smear-negative pulmonary tuberculosis. J Clin Microbiol 41(7):3233–3240

    Article  PubMed  CAS  Google Scholar 

  32. Flamand L, Gravel A, Boutolleau D et al (2008) Multicenter comparison of PCR assays for detection of human herpesvirus 6 DNA in serum. J Clin Microbiol 46(8):2700–2706

    Article  PubMed  CAS  Google Scholar 

  33. Qualtieri J (2009) Stratton CW, Head DR. Tang YW PCR detection of Histoplasma capsulatum var capsulatum in whole blood of a renal transplant patient with disseminated histoplasmosis Ann Clin Lab Sci 39(4):409–412

    CAS  Google Scholar 

  34. Tang YW (2009) Duplex PCR assay simultaneously detecting and differentiating Bartonella quintana, B. henselae, and Coxiella burnetii in surgical heart valve specimens. J Clin Microbiol 47(8):2647–2650

    Article  PubMed  CAS  Google Scholar 

  35. Li H, Dummer JS, Estes WR, Meng S, Wright PF, Tang YW (2003) Measurement of human cytomegalovirus loads by quantitative real-time PCR for monitoring clinical intervention in transplant recipients. J Clin Microbiol 41(1):187–191

    Article  PubMed  CAS  Google Scholar 

  36. Tetzner R (2009) Prevention of PCR cross-contamination by UNG treatment of bisulfite-treated DNA. Methods Mol Biol 507:357–370

    Article  PubMed  CAS  Google Scholar 

  37. Schutten M, Peters D, Back NK et al (2007) Multicenter evaluation of the new Abbott RealTime assays for quantitative detection of human immunodeficiency virus type 1 and hepatitis C virus RNA. J Clin Microbiol 45(6):1712–1717

    Article  PubMed  CAS  Google Scholar 

  38. Leblanc JJ, Pettipas J, Campbell SJ, Davidson RJ, Hatchette TF (2008) Uracil-DNA glycosylase (UNG) influences the melting temperature (T(m)) of herpes simplex virus (HSV) hybridization probes. J Virol Methods 151(1):158–160

    Article  PubMed  CAS  Google Scholar 

  39. Champlot S, Berthelot C, Pruvost M, Bennett EA, Grange T, Geigl EM (2010) An efficient multistrategy DNA decontamination procedure of PCR reagents for hypersensitive PCR applications. PLoS One 5(9)pii:e13042

    Google Scholar 

  40. DeFilippes FM (1991) Decontaminating the polymerase chain reaction. Biotechniques 10(1):26, 8, 30

    Google Scholar 

  41. Isaacs ST, Tessman JW, Metchette KC, Hearst JE, Cimino GD (1991) Post-PCR sterilization: development and application to an HIV-1 diagnostic assay. Nucleic Acids Res 19(1):109–116

    Article  PubMed  CAS  Google Scholar 

  42. Cimino GD, Metchette KC, Tessman JW, Hearst JE, Isaacs ST (1991) Post-PCR sterilization: a method to control carryover contamination for the polymerase chain reaction. Nucleic Acids Res 19(1):99–107

    Article  PubMed  CAS  Google Scholar 

  43. Aslanzadeh J (1993) Application of hydroxylamine hydrochloride for post-PCR sterilization. Mol Cell Probes 7(2):145–150

    Article  PubMed  CAS  Google Scholar 

  44. Woloshynowych M, Rogers S, Taylor-Adams S, Vincent C (2005) The investigation and analysis of critical incidents and adverse events in healthcare. Health Technol Assess 9(19):1–143, iii

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Departments of Pathology and Medicine, Vanderbilt University Medical Center, Nashville, TN, 37212, USA

    Susan Sefers & Charles W. Stratton

  2. Department of Laboratory Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY, 37212, USA

    Yi-Wei Tang

Authors
  1. Susan Sefers
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Charles W. Stratton
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yi-Wei Tang
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Susan Sefers .

Editor information

Editors and Affiliations

  1. Department of Laboratory Medicine, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, 10065, New York, USA

    Yi-Wei Tang

  2. Vanderbilt Clinic, Clinical Microbiology Laboratory, Vanderbilt University Medical Center, 22nd Avenue 1301, Nashville, 37232-5310, Tennessee, USA

    Charles W. Stratton

Rights and permissions

Reprints and permissions

Copyright information

© 2013 Springer Science+Business Media New York

About this chapter

Cite this chapter

Sefers, S., Stratton, C.W., Tang, YW. (2013). Amplification Product Inactivation. In: Tang, YW., Stratton, C. (eds) Advanced Techniques in Diagnostic Microbiology. Springer, Boston, MA. https://doi.org/10.1007/978-1-4614-3970-7_26

Download citation

Publish with us

Policies and ethics

Search

Navigation