Advertisement

Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

False-Positive Results and Contamination in Nucleic Acid Amplification Assays: Suggestions for a Prevent and Destroy Strategy

Abstract

Contamination of samples with DNA is still a major problem in microbiology laboratories, despite the wide acceptance of PCR and other amplification techniques for the detection of frequently low amounts of target DNA. This review focuses on the implications of contamination in the diagnosis and research of infectious diseases, possible sources of contaminants, strategies for prevention and destruction, and quality control. Contamination of samples in diagnostic PCR can have far-reaching consequences for patients, as illustrated by several examples in this review. Furthermore, it appears that the (sometimes very unexpected) sources of contaminants are diverse (including water, reagents, disposables, sample carry over, and amplicon), and contaminants can also be introduced by unrelated activities in neighboring laboratories. Therefore, lack of communication between researchers using the same laboratory space can be considered a risk factor. Only a very limited number of multicenter quality control studies have been published so far, but these showed false-positive rates of 9–57%. The overall conclusion is that although nucleic acid amplification assays are basically useful both in research and in the clinic, their accuracy depends on awareness of risk factors and the proper use of procedures for the prevention of nucleic acid contamination. The discussion of prevention and destruction strategies included in this review may serve as a guide to help improve laboratory practices and reduce the number of false-positive amplification results.

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

References

  1. 1.

    Saiki RK, Scharf S, Faloona F, Mullis KB, Horn GT, Erlich HA, Arnheim N (1985) Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230:1350–1354

  2. 2.

    Compton J (1991) Nucleic acid sequence-based amplification. Nature 350:91–92

  3. 3.

    Wu DY, Wallace RB (1989) The ligation amplification reaction (LAR)-amplification of specific DNA sequences using sequential rounds of template-dependent ligation. Genomics 4:560–569

  4. 4.

    Kwoh DY, Davis GR, Whitfield KM, Chappelle HL, DiMichele LJ, Gingeras TR (1989) Transcription-based amplification system and detection of amplified human immunodeficiency virus type 1 with a bead-based sandwich hybridization format. Proc Nat Acad Sci USA 86:1173–1177

  5. 5.

    Urdea MS (1994) Branched DNA signal amplification. Biotechnol (NY). 12:926–928

  6. 6.

    Walker GT, Fraiser MS, Schram JL, Little MC, Nadeau JG, Malinowski DP (1992) Strand displacement amplification—an isothermal, in vitro DNA amplification technique. Nucleic Acids Res 20:1691–1696

  7. 7.

    Lo YM, Mehal, WZ, Fleming KA (1988) False-positive results and the polymerase chain reaction. Lancet 2:679

  8. 8.

    Millar BC, Xu J, Moore JE (2002) Risk assessment models and contamination management: implications for broad-range ribosomal DNA PCR as a diagnostic tool in medical bacteriology. J Clin Microbiol 40:1575–1580

  9. 9.

    Boyd AS, Annarella M, Rapini RP, Adler-Storthz K, Duvic M (1996) False-positive polymerase chain reaction results for human papillomavirus in lichen planus. Potential laboratory pitfalls of this procedure. J Am Acad Dermatol 35:42–46

  10. 10.

    Schwartz DH, Laeyendecker OB, Arango-Jaramillo S, Castillo RC, Reynolds MJ (1997) Extensive evaluation of a seronegative participant in an HIV-1 vaccine trial as a result of false-positive PCR. Lancet 350:256–259

  11. 11.

    Trinker M, Hofler G, Sill H (1996) False-positive diagnosis of tuberculosis with PCR. Lancet 348:1388

  12. 12.

    Patel R, Grogg KL, Edwards WD, Wright AJ, Schwenk NM (2000) Death from inappropriate therapy for Lyme disease. Clin Infect Dis 31:1107–1109

  13. 13.

    Molloy PJ, Persing DH, Berardi VP (2001) False-positive results of PCR testing for Lyme disease. Clin Infect Dis 33:412–413

  14. 14.

    Landry ML (1995) False-positive polymerase chain reaction results in the diagnosis of herpes simplex encephalitis. J Infect Dis 172:1641–1643

  15. 15.

    Taranger J, Trollfors B, Lind L, Zackrisson G, Beling-Holmquist K (1994) Environmental contamination leading to false-positive polymerase chain reaction for pertussis. Ped Infect Dis J 13:936–937

  16. 16.

    Jehuda-Cohen T (1995) The false-positive polymerase chain reaction and the ostrich. J Infect Dis 172:1420–1421

  17. 17.

    Maksymiuk AW, Thongprasert S, Hopfer R, Luna M, Fainstein V, Bodey GP (1984) Systemic candidiasis in cancer patients. Am J Med 77:20–27

  18. 18.

    Borst A, Leverstein-Van Hall MA, Verhoef J, Fluit AC (2001) Detection of Candida spp. in blood cultures using nucleic acid sequence-based amplification (NASBA). Diagn Microbiol Infect Dis 39:155–160

  19. 19.

    Rimek D, Garg AP, Haas WH, Kappe R (1999) Identification of contaminating fungal DNA sequences in zymolyase. J Clin Microbiol 37:830–831

  20. 20.

    Loeffler J, Hebart H, Bialek R, Hagmeyer L, Schmidt D, Serey FP, Hartmann M, Eucker J, Einsele H (1999) Contaminations occurring in fungal PCR assays. J Clin Microbiol 37:1200–1202

  21. 21.

    Goldenberger D, Altwegg M (1995) Eubacterial PCR: contaminating DNA in primer preparations and its elimination by UV light. J Microbiol Methods 21:27–32

  22. 22.

    Bottger EC (2000) Frequent contamination of Taq polymerase with DNA. Clin Chem 36:1258–1259

  23. 23.

    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 38:1747–1752

  24. 24.

    Hughes MS, Beck LA, Skuce RA (1994) Identification and elimination of DNA sequences in Taq DNA polymerase. J Clin Microbiol 32:2007–2008

  25. 25.

    Maiwald M, Ditton HJ, Sonntag HG, von Knebel DM (1994) Characterization of contaminating DNA in Taq polymerase which occurs during amplification with a primer set for Legionella 5S ribosomal RNA. Mol Cell Probes 8:11–14

  26. 26.

    Rand KH, Houck H (1990) Taq polymerase contains bacterial DNA of unknown origin. Mol Cell Probes 4:445–450

  27. 27.

    Schmidt TM, Pace B, Pace NR (1991) Detection of DNA contamination in Taq polymerase. Biotechniques 11:176–177

  28. 28.

    Van der Zee A, Peeters M, Jong C de, Verbakel H, Crielaard JW, Claas ECJ, Templeton K (2002) Qiagen DNA extraction kits for sample preparation for Legionella PCR are not suitable for diagnostic purposes. J Clin Microbiol 40:1126

  29. 29.

    Kaul K, Luke S, McGurn C, Snowden N, Monti C, Fry WA (1996) Amplification of residual DNA sequences in sterile bronchoscopes leading to false-positive PCR results. J Clin Microbiol 34:1949–1951

  30. 30.

    Schmidt T, Hummel S, Herrmann B (1995) Evidence of contamination in PCR laboratory disposables. Naturwissenschaften 82:423–431

  31. 31.

    Porter-Jordan K, Garrett CT (1990) Source of contamination in polymerase chain reaction assay. Lancet 335:1220

  32. 32.

    Hauman JH, Van Helden PD, Hauman CH (1995) Evacuated tubes and possible false-positive PCR results with blood samples. South Afr Med J 85:119

  33. 33.

    Hartley JL, Rashtchian A (1993) Dealing with contamination: enzymatic control of carryover contamination in PCR. PCR Methods Appl 3 (Suppl):10–14

  34. 34.

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

  35. 35.

    Persing DH (1991) Polymerase chain reaction: trenches to benches. J Clin Microbiol 29:1281–1285

  36. 36.

    Padua RA, Parrado A, Larghero J, Chomienne C (1999) UV and clean air result in contamination-free PCR. Leukemia 13:1898–1899

  37. 37.

    Meier A, Persing DH, Finken M, Bottger EC (1993) Elimination of contaminating DNA within polymerase chain reaction reagents: implications for a general approach to detection of uncultured pathogens. J Clin Microbiol 31:646–652

  38. 38.

    Peters R (1992) Elimination of PCR carryover. Am Biotech Lab 10:42

  39. 39.

    Kitchin PA, Szotyori Z, Fromholc C, Almond N (1990) Avoidance of PCR false positives. Nature 344:201

  40. 40.

    Victor T, Jordaan A, du Toit R, Van Helden PD (1993) Laboratory experience and guidelines for avoiding false positive polymerase chain reaction results. Eur J Clin Chem Clin Biochem 31:531–535

  41. 41.

    Cone RW, Hobson AC, Huang ML, Fairfax MR (1990) Polymerase chain reaction decontamination: the wipe test. Lancet 336:686–687

  42. 42.

    McCormack JM, Sherman ML, Maurer DH (1997) Quality control for DNA contamination in laboratories using PCR-based class II HLA typing methods. Hum Immunol 54:82–88

  43. 43.

    Carroll NM, Adamson P, Okhravi N (1999) Elimination of bacterial DNA from Taq DNA polymerases by restriction endonuclease digestion. J Clin Microbiol 37:3402–3404

  44. 44.

    DeFilippes FM (1991) Decontaminating the polymerase chain reaction. Biotechniques 10:26–30

  45. 45.

    Furrer B, Candrian U, Wieland P, Luthy J (1990) Improving PCR efficiency. Nature 346:324

  46. 46.

    Rochelle PA, Weightman AJ, Fry JC (1992) DNase I treatment of Taq DNA polymerase for complete PCR decontamination. Biotechniques 13:520

  47. 47.

    Widjojoatmodjo MN (1995) Sample preparation for the detection of bacteria in blood using the polymerase chain reaction. In: Diagnosis of infections based on DNA amplification. PhD thesis, Utrecht University, The Netherlands, pp 65–72

  48. 48.

    Widjojoatmodjo MN, Fluit AC, Verhoef J (1994) Rapid identification of bacteria by PCR-single-strand conformation polymorphism. J Clin Microbiol 32:3002–3007

  49. 49.

    Zhu YS, Isaacs ST, Cimino CD, Hearst JE (1991) The use of exonuclease III for polymerase chain reaction sterilization. Nucleic Acids Res 19:251

  50. 50.

    Wages JM Jr, Cai D, Fowler AK (1994) Removal of contaminating DNA from PCR agents by ultrafiltration. Biotechniques 16:1014–1017

  51. 51.

    Dwyer DE, Saksena N (1992) Failure of ultra-violet irradiation and autoclaving to eliminate PCR contamination. Mol Cell Probes 6:87–88

  52. 52.

    Desai UJ, Pfaffle PK (1995) Single-step purification of a thermostable DNA polymerase expressed in Escherichia coli. Biotechniques 19:780–782, 784

  53. 53.

    Engelke DR, Krikos A, Bruck ME, Ginsburg D (1990) Purification of Thermus aquaticus DNA polymerase expressed in Escherichia coli. Anal Biochem 191:396–400

  54. 54.

    Louwrier A (1999) Nucleic acid removal from Taq polymerase preparations using an aqueous/organic biphasic system. Biotechniques 27:444–445

  55. 55.

    Cimino GD, Metchette K, Isaacs ST, Zhu YS (1990) More false-positive problems. Nature 345:773–774

  56. 56.

    Fox JC, Ait-Khaled M, Webster A, Emery VC (1991) Eliminating PCR contamination: is UV irradiation the answer? J Virol Methods 33:375–382

  57. 57.

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

  58. 58.

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

  59. 59.

    Sarkar G, Sommer SS (1990) Shedding light on PCR contamination. Nature 343:27

  60. 60.

    Sarkar G, Sommer SS (1991) Parameters affecting susceptibility of PCR contamination to UV inactivation. Biotechniques 10:590–594

  61. 61.

    Sarkar G, Sommer SS (1993) Removal of DNA contamination in polymerase chain reaction reagents by ultraviolet irradiation. Methods Enzymol 218:381–388

  62. 62.

    Bottema CD, Koeberl DD, Ketterling RP, Bowie EJ, Taylor SA, Lillicrap D, Shapiro A, Gilchrist G, Sommer SS (1990) A past mutation at isoleucine 397 is now a common cause of moderate/mild haemophilia B. Brit J Haematol 75:212–216

  63. 63.

    Gordon LK, Haseltine WA (1982) Quantitation of cyclobutane pyrimidine dimer formation in double- and single-stranded DNA fragments of defined sequence. Rad Res 89:99–112

  64. 64.

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

  65. 65.

    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 19:109–116

  66. 66.

    Niederhauser C, Hofelein C, Wegmuller B, Luthy J, Candrian U (1994) Reliability of PCR decontamination systems. PCR Methods Appl 4:117–123

  67. 67.

    Linquist V, Stoddart CA, McCune JM (1998) UV irradiation of polystyrene pipets releases PCR inhibitors. Biotechniques 24:50–52

  68. 68.

    Cone RW, Fairfax MR (1993) Protocol for ultraviolet irradiation of surfaces to reduce PCR contamination. PCR Methods Appl 3 (Suppl):15–17

  69. 69.

    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 19:99–107

  70. 70.

    Jinno Y, Yoshiura K, Niikawa N (1990) Use of psoralen as extinguisher of contaminated DNA in PCR. Nucleic Acids Res 18:6739

  71. 71.

    Keum WK, Park CE, Lee JH, Khil LY, Kang I, Kim SS, Jung JC, Oh SM, Woo HJ, Lee JH, Kim YC, Yoon Y, Choi JW, Ha J (1997) Primers determine the sensitivity of PCR-mediated hepatitis B virus DNA detection and pretreatment of PCR mixture with 8-methoxypsoralen eliminates false-positive results. Mol Cells 7:244–250

  72. 72.

    Piette J, Hearst J (1985) Sites of termination of in vitro DNA synthesis on psoralen phototreated single-stranded templates. Int J Rad Biol Rel Studies Phys Chem Med 48:381–388

  73. 73.

    Piette JG, Hearst JE (1983) Termination sites of the in vitro nick-translation reaction on DNA that had photoreacted with psoralen. Proc Natl Acad Sci USA 80:5540–5544

  74. 74.

    Shi YB, Gamper H, Hearst JE (1987) The effects of covalent additions of a psoralen on transcription by E. coli RNA polymerase. Nucleic Acids Res 15:6843–6854

  75. 75.

    Deragon JM, Sinnett D, Mitchell G, Potier M, Labuda D (1990) Use of gamma irradiation to eliminate DNA contamination for PCR. Nucleic Acids Res 18:6149

  76. 76.

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

  77. 77.

    Walder RY, Hayes JR, Walder JA (1993) Use of PCR primers containing a 3′-terminal ribose residue to prevent cross-contamination of amplified sequences. Nucleic Acids Res 21:4339–4343

  78. 78.

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

  79. 79.

    Pang J, Modlin J, Yolken R (1992) Use of modified nucleotides and uracil-DNA glycosylase (UNG) for the control of contamination in the PCR-based amplification of RNA. Mol Cell Probes 6:251–256

  80. 80.

    Thornton CG, Hartley JL, Rashtchian A (1992) Utilizing uracil DNA glycosylase to control carryover contamination in PCR: characterization of residual UDG activity following thermal cycling. Biotechniques 13:180–184

  81. 81.

    Bodnar JW, Zempsky W, Warder D, Bergson C, Ward DC (1983) Effect of nucleotide analogs on the cleavage of DNA by the restriction enzymes AluI, DdeI, HinfI, RsaI, and TaqI. J Biol Chem 258:15206–15213

  82. 82.

    Beebe RL, Thornton CG, Hartley JL, Rashtchian A (1992) Contamination-free polymerase chain reaction: endonuclease cleavage and cloning of dU-PCR products. Focus 14:53–56

  83. 83.

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

  84. 84.

    Udaykumar J, Epstein S, Hewlett IK (1993) A novel method employing UNG to avoid carry-over contamination in RNA-PCR. Nucleic Acids Res 21:3917–3918

  85. 85.

    Urban C, Gruber F, Kundi M, Falkner FG, Dorner F, Hammerle T (2000) A systematic and quantitative analysis of PCR template contamination. J Forensic Sci 45:1307–1311

  86. 86.

    Yap EP, Lo YM, Cooper K, Fleming KA, McGee JO (1992) Exclusion of false-positive PCR viral diagnosis by single-strand conformation polymorphism. Lancet 340:736

  87. 87.

    Balfe P (1992) A statistical method for the detection of false positives and false negatives in microtitre format PCR assays. J Virol Methods 39:69–82

  88. 88.

    Bogard M, Buffet-Janvresse C, Cantaloube JF, Biagini P, Duverlie G, Castelain S, Izopet J, Dubois M, Defer C, Lepot I, Coste J, Marcellin P, Martinot-Peignoux M, Halfon P, Gerolami V, Frangeul L, Pawlotsky JM, Roudot-Thoraval F, Dussaix E, Loiseau P, Ravera N, Lewin P, Lamoril J, Lerable J, Lebon P (1997) GEMHEP multicenter quality control study of PCR detection of GB virus C/hepatitis G virus RNA in serum. J Clin Microbiol 35:3298–3300

  89. 89.

    Noordhoek GT, Embden JD van, Kolk AH (1996) Reliability of nucleic acid amplification for detection of Mycobacterium tuberculosis: an international collaborative quality control study among 30 laboratories. J Clin Microbiol 34:2522–2525

  90. 90.

    Quint WG, Heijtink RA, Schirm J, Gerlich WH, Niesters HG (1995) Reliability of methods for hepatitis B virus DNA detection. J Clin Microbiol 33:225–228

  91. 91.

    Zaaijer HL, Cuypers HT, Reesink HW, Winkel IN, Gerken G, Lelie PN (1993) Reliability of polymerase chain reaction for detection of hepatitis C virus. Lancet 341:722–724

Download references

Author information

Correspondence to A. C. Fluit.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Borst, A., Box, A.T.A. & Fluit, A.C. False-Positive Results and Contamination in Nucleic Acid Amplification Assays: Suggestions for a Prevent and Destroy Strategy. Eur J Clin Microbiol Infect Dis 23, 289–299 (2004). https://doi.org/10.1007/s10096-004-1100-1

Download citation

Keywords

  • Polymerase Chain Reaction
  • Human Papilloma Virus
  • Polymerase Chain Reaction Assay
  • Lyme Disease
  • Psoralen