Three distinct levels of quality control should be considered when setting up PCR applications for use in the clinical laboratory. The primary level of quality control involves the organization of the laboratory and relevant logistical aspects, which are areas of major importance with respect to the avoidance of PCR contamination and the prevention of false positive clinical results. The secondary level of quality control involves PCR design, optimization and assay quality control per se. Finally, there may be individual quality considerations that are specifically related to individual types of PCR methodologies (especially reverse transcription PCR methodologies).
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsPreview
Unable to display preview. Download preview PDF.
References
Al Soud WA, Radstrom P. 1998. Capacity of nine thermostable DNA polymerases to mediate amplification in the presence of PCR inhibiting substances. J Environ Microbiol 64:3748–3753.
Al Soud WA, Radstrom P. 2000. Effects of amplification facilitators on diagnostic PCR in the presence of blood, feces and meat. J Clin Microbiol 38:4463–4470.
Al Soud WA, Radstrom P. 2001. Purification and characterization of PCR-inhibitory components in blood cells. J Clin Microbiol 39(2):485–493.
Baldrick E, Xamena N, Cabre O. 1999. Overcoming false negatives due to the genomic context in PCR amplification. J Biochem Biophys Methods 40:45–48.
Belec L, Authier J, Eliezer-Vanerot MC, Piedouillet C, Mohamed AS, Gherardi RK. 1998. Myoglobin as a PCR inhibitor: a limitation for PCR from skeletal muscle tissue avoided by the use of Thermus thermophilus polymerase. Muscle Nerve 21:1064–1067.
Buffone GJ, Demmler GJ, Schimbor CM, Greer J. 1991. Improved amplification of CMV DNA from urine after purification of DNA with glass beads. Clin Chem 37:1945–1949.
Cobb BD, Clarkson JM. 1994. A simple procedure for optimizing the PCR using modified Taguchi methods. Nucleic Acids Res 22:3801–3805.
Dakhama A, Maeck V, Hogg JC, Hegele RG. 1996. Amplification of human beta-actin gene by the reverse transcriptase PCR: implications for assessment of RNA from formalin fixed paraffin embedded material. J Histochem Cytochem 44:1205–1207.
Finke J, Fritzen R, Ternes P, Lange W, Dolken G. 1993. An improved strategy and a useful housekeeping gene for RNA analysis from formalin fixed paraffin embedded tissues by PCR. Biotechniques 14:448–453.
Foss AJ, Guille MJ, Occleston NL, Hykin PG, Hungerford JL, Lightman S. 1995. The detection of melanoma cells in peripheral blood by reverse transcription PCR. Br J Cancer 72:155–159.
Golenberg EM, Bickel A, Weihs P. 1996. Effect of highly fragmented DNA on PCR. Nucleic Acids Res 24:5026–5033.
Greer CE. 1991. PCR amplification from paraffin-embedded tissues: effect of fixative and fixation time. Am J Clin Pathol 95:117–124.
Jinno Y, Yoshiura K, Niikawa N. 1990. Use of psoralen as extinguisher of contaminated DNA in PCR. Nucleic Acids Res 18:6739.
Kang J, Lee MS, Gorenstein DG. 2005. The enhancement of PCR amplification of a random sequence DNA library by DMSO and betaine: application to in vitro combinatorial selection of aptamers. J Biochem Biophys Methods 64(2):147–151.
KlaschikS, Lehman LE, Raadts A, Book M, Hoeft A, Stuber F. 1999. Comparison of different decontamination methods for reagents to detect low concentrations of bacterial 16S DNA by real-time PCR. Mol Biotechnol 3:231–242.
Kleter B, Van Doorn LJ, Ter Schegget J, Schrauwen L, Van Krimpen K, Burger M, Ter Harmsel B, Quint W. 1998. Novel short fragment PCR assay for highly sensitive broad-spectrum detection of anogenital human papillomaviruses. Am J Pathol 153:1731–1739.
Kofler B, Klausegger A. 1999. Simplified PCR set-up using a frozen preformulated mix for the detection of CMV. Diagn Microbiol Infect Dis 34:33–35.
Koopmans M, Monroe SS, Coffield LM, Zaki SR. 1993. Optimization of extraction and PCR amplification of RNA extracts from paraffin embedded tissue in different fixatives. J Virol Methods 43:189–204.
Kunakorn M, Raksakai M, Niemhorn S, Chongtrakool P, Pracharktam R. 2002. Overcoming the errors of in-house PCR used in the clinical laboratory for the diagnosis of extra-pulmonary tuberculosis. Southeast Asian J Trop Med Public Health 30:84–90.
Lewis SM. 1995. Quality assurance programmes in the United Kingdom. Ann Ist Super Sanita 31:53–59.
Linssen B, Kinney RM, Aguilar PM, Russell KL, Watts DM, Kaaden O, Pfeffer M. 2000. Development of reverse transcription PCR assays specific for the detection of equine encephalitis viruses. J Clin Microbiol 38:1527–1535.
Matthews JL, Chung M, Matyas RJ. 2002. Persistent DNA contamination in competitive RT-PCR using cRNA internal standards: identity, quantity and control. Biotechniques 32:1412–1417.
Noordhoek GT, Van Embden JD, Kolk AH. 1996. Reliability of nucleic acid amplification for detection of Mycobacterium tuberculosis: and international collaborative quality control study among 30 laboratories. J Clin Microbiol 34:2522–2525.
Ou CY, Moore JL, Schochetman G. 1991. Use of UV irradiation to reduce false positivity in PCR. Biotechniques 10:442–446.
Radonic A, Thulke S, Mackai TM. 2004. Guideline for reference gene selection for quantitative real-time PCR. Biol Bioch Res Commun 313:856–862.
Rees WA, Yager TD, Korte J, Von Hippel PH. 1993. Betaine can eliminate the base pair composition dependence of DNA melting. Biochem 32:137–144.
Sellon RK. 2003. Update on molecular techniques for diagnostic testing of infectious diseases. Vet Clin North Am Small Anim Pract 33:677–693.
Shlyakhtenko LS, Potaman VN, Sinden RR, Lyubchenko YL. 1998. Structure and dynamics of supercoil stabilized DNA cruciforms. J Mol Biol 280:61–72.
Sirko DA, Ehrlich GD. 1994. PCR laboratory facilities, protocols and operations. In: Ehrlich GD, Greenberg SJ (eds) PCR-based diagnostics in infectious disease. Blackwell, Boston, MA, pp. 19–44.
Sonneveld T, Tobutt KR, Robbins TP. 2003. Allele specific PCR detection of sweet cherry self incompatibility (S) alleles S1 to S16 using consensus and allele specific primers. Theor Appl Genet 107:1059–1070.
Soukup J, Krskova L, Hilska I, Kodet R. 2003. Ethanol fixation of lymphoma samples as an alternative approach for preservation of the nucleic acids. Neoplasma 50:300–304.
Tournier I, Bernuau D, Poliard A, Schoevaert D, Feldmann G. 1987. Detection of albumin mRNAs in rat liver by in situ hybridization: usefulness of paraffin embedding and comparison of various fixation procedures. J Histochem Cytochem 35:453–459.
Tzanakaki G, Tsolia M, Vlachou V, Theodoridou M, Pangalis A, Foustoukou M, Karpathios T, Blackwell CC, Kremastinou J. 2003. Evaluation of non-culture diagnosis of invasive meningococcal disease by PCR. FEMS Immunol Med Microbiol 39:31–36.
Van Belkum A, Scherer S, Van Alphen L, Verbrugh H. 1998. Short sequence DNA repeats in prokaryotic genomes. Microbiol Mol Biol Rev 62:275–293.
Van den Brule A, Claas ECJ, Du Maine M, Melchers WJ, Helmerhorst T, Quint WG, Lindeman J, Meijer CJ, Walboomers J. 1989. Use of anti-contamination primers in the PCR for the detection of human papillomavirus genotypes in cervical scrapes and biopsies. J Med Virol 29:20–27.
Wallace PS. 2003. Linkage between the journal and the Quality Control Molecular Diagnostics (QCMD). J Clin Virol 27:211–212.
Wiedbrauk DL, Werner JC, Drevon AM. 1995. Inhibition of PCR by aqueous and vitreous fluids. J Clin Microbiol 33:2643–2646.
Rights and permissions
Copyright information
© 2008 Springer Science + Business Media B.V
About this chapter
Cite this chapter
(2008). Ensuring PCR Quality – Laboratory Organisation, PCR Optimization and Controls. In: Principles and Technical Aspects of PCR Amplification. Springer, Dordrecht. https://doi.org/10.1007/978-1-4020-6241-4_10
Download citation
DOI: https://doi.org/10.1007/978-1-4020-6241-4_10
Publisher Name: Springer, Dordrecht
Print ISBN: 978-1-4020-6240-7
Online ISBN: 978-1-4020-6241-4
eBook Packages: Biomedical and Life SciencesBiomedical and Life Sciences (R0)