Abstract
Polymerase chain reaction (PCR) is recognized as a rapid, sensitive, and specific molecular diagnostic tool for the analysis of nucleic acids. However, the sensitivity and kinetics of diagnostic PCR may be dramatically reduced when applied directly to biological samples, such as blood and feces, owing to PCR-inhibitory components. As a result, pre-PCR processing procedures have been developed to remove or reduce the effects of PCR inhibitors. Pre-PCR processing comprises all steps prior to the detection of PCR products, that is, sampling, sample preparation, and deoxyribonucleic acid (DNA) amplification. The aim of pre-PCR processing is to convert a complex biological sample with its target nucleic acids/cells into PCR-amplifiable samples by combining sample preparation and amplification conditions. Several different pre-PCR processing strategies are used: (1) optimization of the DNA amplification conditions by the use of alternative DNA polymerases and/or amplification facilitators, (2) optimization of the sample preparation method, (3) optimization of the sampling method, and (4) combinations of the different strategies. This review describes different pre-PCR processing strategies to circumvent PCR inhibition to allow accurate and precise DNA amplification.
Similar content being viewed by others
References
Lantz, P. G., Abu Al-Soud, W., Knutsson, R., Hahn-Hägerdal, B., and Rådström, P. (2000) Biotechnical use of the polymerase chain reaction for microbiological analysis of biological samples. Biotechnol. Annu. Rev. 5, 87–130.
Abu Al-Soud, W. and Rådström, P. (1998) Capacity of nine thermostable DNA polymerases to mediate DNA amplification in the presence of PCR-inhibiting samples. Appl. Environ. Microbiol. 64, 3748–3753.
Rossen, L., Nørskov, P., Holmstrøm, K., and Rasmussen, O. F. (1992) Inhibition of PCR by components of food samples, microbial diagnostic assays and DNA-extraction solutions. Int. J. Food Microbiol. 17, 37–45.
Wilson, I. G. (1997) Inhibition and facilitation of nucleic acid amplification. Appl. Environ. Microbiol. 63, 3741–3751.
Abu Al-Sound, W. and Rådström, P. (2001) Purification and characterization of PCR-inhibitory components in blood cells. J. Clin. Microbiol. 39, 485–493.
Lantz, P.-G., Matsson, M., Wadström, T., and Rådström, P. (1997) Removal of PCR inhibitors from human faecal samples through the use of an aqueous two-phase system for sample preparation prior to PCR. J. Microbiol. Methods 28, 159–167.
Onteiro, L., Bonnemaison, D., Vekris, A., et al. (1997) Complex polysaccharides as PCR inhibitors in feces: Helicobacter pylori model. J. Clin. Microbiol. 35, 995–998.
Kim, C. H., Khan, M., Morin, D. E., et al. (2001) Optimization of the PCR for detection of Staphylococcus aureus nuc gene in bovine milk. J. Dairy Sci. 84, 74–83.
Akane, A., Matsubara, K., Nakamura, H., Takahashi, S., and Kimura, K. (1994) Identification of the heme compound copurified with deoxyribonucleic acid (DNA) from bloodstains, a major inhibitor of polymerase chain reaction (PCR) amplification. J. Forensic Sci. 39, 362–372.
Tsai, Y. L. and Olson, B. H. (1992) Rapid method for separation of bacterial DNA from humic substances in sediments for polymerase chain reaction. Appl. Environ. Microbiol. 58, 2292–2295.
Eckhart, L., Bach, J., and Tschachler, E.. (2000) Melanin binds reversibly to thermostable DNA polymerase and inhibits its activity. Biochem. Biophys. Res. Commun. 271, 726–730.
Belec, L., Authier, J., Eliezer-Vanerot, M. C., Piedouillet, C., Mohamed, A. S., and Gherardi, R. K. (1998) Myoglobin as a polymerase chain reaction (PCR) inhibitor: a limitation for PCR from skeletal muscle tissue avoided by the use of Thermus thermophilus polymerase. Muscle Nerve 21, 1064–1067.
Demeke, T. and Adams, R. P. (1992) The effects of plant polysaccharides and buffer additives on PCR. Biotechniques 12, 332–334.
Bickley, J., Short, J. K., McDowel, D. G., and Parkes, H. C. (1996) Polymerase chain reaction (PCR) detection of Listeria monocytogenes in diluted milk and reversal of PCR inhibition caused by calcium ions. Lett. Appl. Microbiol. 22, 153–158.
Powell, H. A., Gooding, C. M., Garret, S. D., Lund, B. M., and McKee, R. A. (1994) Proteinase inhibition of the detection of Listeria monocytogenes in milk using the polymerase chain reaction. Lett. Appl. Microbiol. 18, 59–61.
Khan, G., Kangro, H. O., Coates, P. J., and Heath, R. B. (1991) Inhibitory effects of urine on the polymerase chain reaction for cytomegalovirus DNA. J. Clin. Pathol. 44, 360–365.
Abu Al-Soud, W., Jönsson, L. J., and Rådström, P. (2000) Identification and characterization of immunoglobulin G in blood as a major inhibitor of diagnostic PCR. J. Clin. Microbiol. 38, 345–350.
Byrnes, J. J., Downey, K. M., Esserman, L., and So, A. G. (1975) Mechanism of hemin inhibition of erythroid cytoplasmic DNA polymerase. Biochemistry 14, 796–799.
Jaffe, R. I., Lane, J. D., and Bates, C. W. (2001) Realtime identification of Pseudomonas aeruginosa direct from clinical samples using a rapid extraction method and polymerase chain reaction (PCR). J. Clin. Lab. Anal. 15, 131–137.
Bailey, J. S. (1998) Detection of Salmonella cells within 24 to 26 hours in poultry samples with the polymerase chain reaction BAX system. J. Food Prot. 61, 792–795.
Dahlenborg, M., Borch, E., and Rådström, P. (2001) Development of a combined selection and enrichment PCR procedure for Clostridium botulinum types B, E, and F and its use to determine prevalence in fecal samples from slaughtered pigs. Appl. Environ. Microbiol. 67, 4781–4788.
Fahle, G. A. and Fischer, S. H. (2000) Comparison of six commercial DNA extraction kits for recovery of cytomegalovirus DNA from spiked human specimens. J. Clin. Microbiol. 38, 3860–3863.
Freise, J., Gerard, H. C., Bunke, T., et al. (2001) Optimised sample DNA preparation for detection of Chlamydia trachomatis in synovial tissue by polymerase chain reaction and ligase chain reaction. Ann. Rheum. Dis. 60, 140–145.
Lantz, P. G., Knutsson, R., Blixt, Y., Al Soud, W. A., Borch, E., and Rådström, P. (1998) Detection of pathogenic Yersinia enterocolitica in enrichment media and pork by a multiplex PCR: a study of sample preparation and PCR-inhibitory components. Int. J. Food Microbiol. 45, 93–105.
Shafer, R. W., Levee, D. J., Winters, M. A., Richmond, K. L., Huang, D., and Merigan, T. C. (1997) Comparison of QIAamp HCV kit spin columns, silica beads, and phenol-chloroform for recovering human immunodeficiency virus type 1 RNA from plasma. J. Clin. Microbiol. 35, 520–522.
Kramvis, A., Bukofzer, S., and Kew, M. C. (1996) Comparison of hepatitis B virus DNA extractions from serum by the QIAamp blood kit, GeneReleaser, and the phenol-chloroform method. J. Clin. Microbiol. 34, 2731–2733.
Hallier-Soulier, S. and Guillot, E. (1999) An immunomagnetic separation polymerase chain reaction assay for rapid and ultra-sensitive detection of Cryptosporidium parvum in drinking water. FEMS Microbiol. Lett. 176, 285–289.
Antognoli, M. C., Salman, M. D., Triantis, J., Hernandez, J., and Keefe, T. (2001) A one-tube nested polymerase chain reaction for the detection of Mycobacterium bovis in spiked milk samples: an evaluation of concentration and lytic techniques. J. Vet. Diagn. Invest. 13, 111–116.
Jothikumar, N., Cliver, D. O., and Mariam, T. W. (1998) Immunomagnetic capture PCR for rapid concentration and detection of hepatitis A virus from environmental samples. Appl. Environ. Microbiol. 64, 504–508.
Lantz, P.-G., Tjerneld, F., Hahn-Hägerdal, B., and Rådström, P. (1996) Use of aqueous two-phase systems in sample preparation for polymerase chain reaction-based detection of microorganisms. J. Chromat. B Biomed. Appl. 680, 165–170.
Lindqvist, R., Norling, B., and Thisted Lambertz, S. (1997) A rapid sample preparation method for PCR detection of food pathogens based on buoyant density centrifugation. Lett. Appl. Microbiol. 24, 306–310.
Gerritsen, M. J., Olyhoek, T., Smits, M. A., and Bokhout, B. A. (1991) Sample preparation method for polymerase chain reaction-based semiquantitative detection of Leptospira interrogans serovar hardjo subtype hardjobovis in bovine urine. J. Clin. Microbiol. 29, 2805–2808.
Starbuck, M. A., Hill, P. J., and Stewart, G. S. (1992) Ultra sensitive detection of Listeria monocytogenes in milk by the polymerase chain reaction (PCR). Lett. Appl. Microbiol. 15, 248–252.
Abu Al-Soud, W., Lantz, P.-G., Bäckman, A., Olcén, P., and Rådström, P. (1998) A sample preparation method which facilitates detection of bacteria in blood cultures by the polymerase chain reaction. J. Microbiol. Methods 32, 217–224.
Lindqvist, R. (1997) Preparation of PCR samples from food by a rapid and simple centrifugation technique evaluated by detection of Escherichia coli O157:H7. Int. J. Food Microbiol. 37, 73–82.
Thisted Lambertz, S., Lindqvist, R., Ballagi-Pordány, A., and Danielsson-Tham, M.-L. (2000) A combined culture and PCR method for detection of pathogenic Yersinia enterocolitica in food. Int. J. Food Microbiol. 57, 63–73.
Sharma, V. K. and Carlson, S. A. (2000) Simultaneous detection of Salmonella strains and Escherichia coli O157:H7 with fluorogenic PCR and single-enrichment-broth culture. Appl. Environ. Microbiol. 66, 5472–5476.
Knutsson, R., Blixt, Y., Grage, H., Borch, E., and Rådström, P. (2002) Evaluation of selective enrichment PCR procedures for Yersinia enterocolitica. Int. J. Food Microbiol. 73, 35–46.
Minton, A. P. and Wilf, J. (1981) Effect of macromolecular crowding upon the structure and function of an enzyme: glyceraldehyde-3-phosphate dehydrogenas. Biochemistry 20, 4821–4826.
Zimmerman, S. B. and Trach, S. O. (1988) Macromolecular crowding extends the range of conditions under which DNA polymerase is functional. Biochim. Biophys. Acta. 949, 297–304.
Saiki, R. K., Scharf, S., Faloona, F., et al. (1985) Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230, 1350–1354.
Saiki, R. K., Gelfand, D. H., Stoffel, S., et al. (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239, 487–491.
Katcher, H. L. and Schwartz, I. (1994) A distinctive property of Tth DNA polymerase: enzymatic amplification in the presence of phenol. Biotechniques 16, 84–92.
Zsolnai, A. and Fesus, L. (1997) Enhancement of PCR-RFLP typing of bovine leukocyte adhesion deficiency. Biotechniques 23, 380–382.
Diakou, A. and Dovas, C. I. (2001) Optimization of random-amplified polymorphic DNA producing amplicons up to 8500 bp and revealing intraspecies polymorphism in Leishmania infantum isolates. Anal. Biochem. 288, 195–200.
Hendolin, P. H., Paulin, L., and Ylikoski, J. (2000) Clinically applicable multiplex PCR for four middle ear pathogens. J. Clin. Microbiol. 38, 125–132.
Haag, E. and Raman, V. (1994) Effects of primer choice and source of Taq DNA polymerase on the banding patterns of differential display RT-PCR. Biotechniques 17, 226–228.
Gál, J., Schnell, R., and Kálmán, M. (2000) Polymerase dependence of autosticky polymerase chain reaction. Anal. Biochem. 282, 156–158.
Tebbe, C. C. and Vahjen, W. (1993) Interference of humic acids and DNA extracted directly from soil in detection and transformation of recombinant DNA from bacteria and a yeast. Appl. Environ. Microbiol. 59, 2657–2665.
Favre, N. and Rudin, W. (1996) Salt-dependent performance variation of DNA polymerases in co-amplification PCR. Biotechniques 21, 28–30.
Wiedbrauk, D. L., Werner, J. C., and Drevon, A. M. (1995) Inhibition of PCR by aqueous and vitreous fluids. J. Clin. Microbiol. 33, 2643–2646.
Poddar, S. K., Sawyer, M. H., and Connor, J. D. (1998) Effect of inhibitors in clinical specimens on Taq and Tth DNA polymerase-based PCR amplification of influenza A virus. J. Med. Microbiol. 47, 1131–1135.
Akalu, A. and Reichardt, J. K. (1999) A reliable PCR amplification method for microdissected tumor cells obtained from paraffin-embedded tissue. Genet. Anal. 15, 229–233.
Panaccio, M. and Lew, A. (1991) PCR based diagnosis in the presence of 8% (v/v) blood. Nucleic Acids Res. 19, 1151.
Kebelmann-Betzing, C., Seeger, K., Dragon, S., et al. (1998) Advantages of a new Taq DNA polymerase in multiplex PCR and time-release PCR. Biotechniques 24, 154–158.
Moretti, T., Koons, B., and Budowle, B. (1998) Enhancement of PCR amplification yield and specificity using AmpliTaq Gold DNA polymerase. Biotechniques 25, 716–722.
Shames, B., Fox, J. G., Dewhirst, F., Yan, L., Shen, Z., and Taylor, N. S. (1995) Identification of wide-spread Helicobacter hepaticus infection in feces in commercial mouse colonies by culture and PCR assay. J. Clin. Microbiol. 33, 2968–2972.
Kainz, P., Schmiedlechner, A., and Strack, H. B. (2000) Specificity-enhanced hot-start PCR: addition of double-stranded DNA fragments adapted to the annealing temperature. Biotechniques 28, 278–282.
Kainz, P. (2000) The PCR plateau phase—towards an understanding of its limitations. Biochim. Biophys. Acta. 1494, 23–27.
Laigret, F., Deaville, J., Bove, J. M., and Bradbury, J. M. (1996) Specific detection of Mycoplasma iowa using polymerase chain reaction. Mol. Cell Probes 10, 23–29.
Pomp, D. and Medrano, J. F. (1991) Organic solvents as facilitators of polymerase chain reaction. Biotechniques 10, 58–59.
Abu Al-Soud, W. (2000) Optimisation of diagnostic PCR: a study of PCR inhibitors in blood and sample pretreatment. Doctoral thesis. Department of Applied Microbiology, Lund University, Lund, Sweden.
Abu Al-Soud, W. and Rådström, P. (2000) Effects of amplification facilitators on diagnostic PCR in the presence of blood, feces, and meat. J. Clin. Microbiol. 38, 4463–4470.
Poussier, S., Cheron, J. J., Couteau, A., and Luisetti, J. (2002) Evaluation of procedures for reliable PCR detection of Ralstonia solanacearum in common natural substrates. J. Microbiol. Methods 51, 349–359.
Kreader, C. A. (1996) Relief of amplification inhibition in PCR with bovine serum albumin or T4 gene 32 protein. Appl. Environ. Microbiol. 62, 1102–1106.
Tsutsui, K. and Mueller, G. C. (1987) Hemin inhibits virion-associated reverse transcriptase of murine leukemia virus. Biochem. Biophys. Res. Commun. 149, 628–634.
Topal, M. D. and Sinha, N. K. (1983) Products of bacteriophage T4 genes 32 and 45 improve the accuracy of DNA replication in vitro. J. Biol. Chem. 258, 12,274–12,279.
Chandler, D. P., Wagnon, C. A., and Bolton, H., Jr. (1998) Reverse transcriptase (RT) inhibition of PCR at low concentrations of template and its implications for quantitative RT-PCR. Appl. Environ. Microbiol. 64, 669–677.
Jordan, S. P., Zugay, J., Darke, P. L., and Kuo, L. C. (1992) Activity and dimerization of human immunodeficiency virus protease as a function of solvent composition and enzyme concentration. J. Biol. Chem. 267, 20,028–20,032.
Wu, J. R. and Yeh, Y. C. (1973) Requirement of a functional gene 32 product of bacteriophage T4 in UV repair. J. Virol. 12, 758–765.
Varadaraj, K. and Skinner, D. M. (1994) Denaturants or cosolvents improve the specificity of PCR amplification of a G + C-rich DNA using genetically engineered DNA polymerases. Gene 140, 1–5.
Lee, C. H., Mizusawa, H., and Kakefuda, T. (1981) Unwinding of double-stranded DNA helix by dehydration. Proc. Natl. Acad. Sci. USA 78, 2838–2842.
Dutton, C. M., Paynton, C., and Sommer, S. S. (1993) General method for amplifying regions of very high G+C content. Nucleic Acids Res. 21, 2953–2954.
Innis, M. A., Myambo, K. B., Gelfand, D. H., and Brow, M. A. (1988) DNA sequencing with Thermus aquaticus DNA polymerase and direct sequencing of polymerase chain reaction-amplified DNA. Proc. Natl. Acad. Sci. USA 85, 9436–9440.
Frackman, S., Kobs, G., Simpson, D., and Storts, D. (1998) Betaine and DMSO: enhancing agents for PCR. Promega Notes 27,
Henke, W., Herdel, K., Jung, K., Schnorr, D., and Loening, S. A. (1997) Betaine improves the PCR amplification of GC-rich DNA sequences. Nucleic Acids Res. 25, 3957–3958.
Back, J. F., Oakenfull, D., and Smith, M. B. (1979) Increased thermal stability of proteins in the presence of sugars and polyols. Biochemistry 18, 5191–5196.
Nagai, M., Yoshida, A., and Sato, N. (1998) Additive effects of bovine serum albumin, dithiothreitol, and glycerol on PCR. Biochem. Mol. Biol. Int. 44, 157–163.
Kim, S., Labbe, R. G., and Ryu, S. (2000) Inhibitory effects of collagen on the PCR for detection of Clostridium perfringens. Appl. Environ. Microbiol. 66, 1213–1215.
Chen, S., Yee, A., Griffiths, M., et al. (1997) The evaluation of a fluorogenic polymerase chain reaction assay for the detection of Salmonella species in food commodities. Int. J. Food. Microbiol. 35, 239–250.
Knutsson, R., Fontanesi, M., Grage, H., and Rådström, P. (2002) Development of a PCR-compatible enrichment medium for Yersinia enterocolitica: amplification precision and dynamic detection range during cultivation. Int. J. Food Microbiol. 72, 185–201.
Grant, K. A., Dickinson, J. H., Payne, M. J., Campbell, S., Collins, M. D., and Kroll, R. G. (1993) Use of the polymerase chain reaction and 16S rRNA sequences for the rapid detection of Brochothrix spp. in foods. J. Appl. Bacteriol. 74, 260–267.
Klein, P. G. and Juneja, V. K. (1997) Sensitive detection of viable Listeria monocytogenes by reverse transcription-PCR. Appl. Environ. Microbiol. 63, 4441–4448.
Nordvåg, B., Riise, H., Husby, G., Nilsen, I., and El-Gewely, M. R. (1995) Direct use of blood in PCR. Methods Neurosci. 26, 15–25.
Seesod, N., Lundeberg, J., Hedrum, A., et al. (1993) Immunomagnetic purification to facilitate DNA diagnosis of Plasmodium falciparum. J. Clin. Microbiol. 31, 2715–2719.
Lantz, P. G., Tjerneld, F., Borch, E., Hahn-Hägerdal, B., and Rådström, P. (1994) Enhanced sensitivity in PCR detection of Listeria monocytogenes in soft cheese through use of an aqueous two-phase system as a sample preparation method. Appl. Environ. Microbiol. 60, 3416–3418.
Giambernardi, T. A., Rodeck, U., and Klebe, R. J. (1998) Bovine serum albumin reverses inhibition of RT-PCR by melanin. Biotechniques 25, 564–566.
Sidhu, M. K., Liao, M. J., and Rashidbaigi, A. (1996) Dimethyl sulfoxide improves RNA amplification. Biotechniques 21, 44–47.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Rådström, P., Knutsson, R., Wolffs, P. et al. Pre-PCR processing. Mol Biotechnol 26, 133–146 (2004). https://doi.org/10.1385/MB:26:2:133
Issue Date:
DOI: https://doi.org/10.1385/MB:26:2:133