False negative results from using common PCR reagents
© O'Keefe et al; licensee BioMed Central Ltd. 2011
Received: 19 July 2011
Accepted: 27 October 2011
Published: 27 October 2011
The sensitivity of the PCR reaction makes it ideal for use when identifying potentially novel viral infections in human disease. Unfortunately, this same sensitivity also leaves this popular technique open to potential contamination with previously amplified PCR products, or "carry-over" contamination. PCR product carry-over contamination can be prevented with uracil-DNA-glycosylase (UNG), and it is for this reason that it is commonly included in many commercial PCR master-mixes. While testing the sensitivity of PCR assays to detect murine DNA contamination in human tissue samples, we inadvertently discovered that the use of this common PCR reagent may lead to the production of false-negative PCR results.
We show here that contamination with minute quantities of UNG-digested PCR product or any negative control PCR reactions containing primer-dimers regardless of UNG presence can completely block amplification from as much as 60 ng of legitimate target DNA.
These findings could potentially explain discrepant results from laboratories attempting to amplify MLV-related viruses including XMRV from human samples, as none of the published reports used internal-tube controls for amplification. The potential for false negative results needs to be considered and carefully controlled in PCR experiments, especially when the target copy number may be low - just as the potential for false positive results already is.
Carry-over contamination can be prevented by incorporating uracil instead of thymine into the PCR product, then treating with uracil-DNA-glycosylase (UNG) prior to initiating subsequent PCR reactions . The UNG degrades DNA containing uracil, thereby degrading contaminating PCR products while leaving non-uracil DNA (ie. target DNA) intact. This method is so effective that several major brands of Taq and Taq master-mixes include this enzyme and replace dTTP with dUTP. In addition the enzyme is sold separately and as part of kits designed to prevent carry-over contamination.
Xenotropic Murine leukemia virus Related Virus (XMRV) was isolated from patients with prostate cancer in 2006, and since that time many reports have either identified XMRV or the highly related murine leukemia viruses (MLVs) in various patient cohorts. However, many reports have been unable to find either of these types of viruses. Mice, particularly strains of mice used in laboratory research, carry 20-40 copies of MLV-type viruses. Therefore MLV-type viral DNA detected in patient samples could be due to actual MLV infection of patient tissue or contamination of patient samples with mouse DNA. We developed an assay to detect femtogram level contamination of murine DNA in human DNA samples. This sensitivity is necessary because MLV-related viruses found in human populations seem to be present at very low levels. Therefore low levels of murine DNA contamination could give rise to false-positive results.
Use of Uracil-DNA-glycosylase inhibits amplification of legitimate target DNA in the presence of low levels of PCR carry-over contamination
Primer-dimers from previously generated reactions can also inhibit legitimate target amplification, regardless of the presence of uracil-DNA-glycosylase
Primer dimers from previously generated reactions demonstrate a strong inhibitory effect on legitimate target amplification of an MLV-carrying plasmid
MLVs previously found to be present in patient samples have been observed at very low levels. Therefore we wanted to examine the inhibitory effect that minute levels of primer-dimer contamination (10-7 dilution) can have on Gag PCR product formation from MLVs that are present at low copy number. Figure 3b demonstrates that contamination of MLV present at 200, 000 copies or less with 10-7 dilution of Gag PCR primer-dimers from a previous PCR reaction almost completely inhibits PCR product formation. This data suggests that contamination with extremely small quantities of primer-dimers generated from previous PCR reactions can inhibit PCR product formation, thus leading to false negative results.
Taken together, these data suggest that inhibition of legitimate target occurs from UNG-digested PCR products or from contamination with primer-dimers from a previous PCR reaction, regardless of the presence of UNG. Thus contamination from any previous PCR samples can result in either false positives (which may or may not be detected in the negative control depending upon the samples that are contaminated and whether UNG is used) or false negatives which will not be detected using standard PCR controls.
Discussion and Conclusions
As these results could potentially explain some of the discrepancies in amplification of MLV-related viruses from different laboratories, we surveyed these publications to attempt to determine if the authors used UNG-containing master-mixes. Of concern, despite the importance of the actual PCR methods used to amplify these potentially novel viruses, we were unable to determine the type of Taq or master-mix used in 11 of 38 publications. Of the remaining 27 publications, 13 studies used Taq or master-mixes likely containing UNG (it was present in 8 studies [2–9] and possibly used in the remaining 5 studies [10–14] that we examined). Therefore contamination at either the individual sample level or in the PCR reaction itself could have inhibited amplification of legitimate target, especially if the target is found at low levels. Additionally, as shown in Figure 2, regardless of the presence of UNG, PCR can be inhibited if there is even a minute amount of contamination from previous (negative) PCR reactions.
Based on our data, we could speculate that low levels of PCR product contamination may not be sufficient to significantly block amplification of the positive control in these reactions, especially if the positive control is the target cloned into a plasmid and present at a high copy number. In the majority of the studies commented on in this manuscript, the positive control used was an XMRV plasmid, and most papers were not clear about the amount of plasmid used for control amplification. Therefore, amplification of the high copy number positive control could occur while samples positive for a low copy number virus could be inhibited by carry-over PCR contamination.
To circumvent the potential for PCR inhibition, a positive control included in each PCR reaction is necessary to confirm that the PCR is not inhibited. To accomplish this, a synthetic target template with identical primer-binding sites but different size could be constructed, and spiked into each test sample. This would result in potential amplification of two bands: one band to detect the target DNA and the other to detect the synthetic target. In addition, the copy number of the synthetic target spiked into the test samples would need to be at the level of detection of the assay, so as any inhibition would result in lack of amplification. None of the 38 MLV-related publications we examined used internal PCR controls. Given the data presented in this manuscript, we would propose that in cases such as the debate regarding the existence of novel infectious viruses, failure to find a virus is not the equivalent of evidence against its existence. The potential for false negative results needs to be considered and carefully controlled in PCR experiments, just as the potential for false positive results already is.
Human LNCaP cell line DNA was extracted with the DNeasy® Blood & Tissue Kit (Qiagen). DNA from mouse tails was extracted with the Wizard SV Genomic DNA purification system (Promega). Approval for mouse work was given by the University of Pittsburgh IACUC, under protocol #1991385A, and the work was performed according to internationally accepted guidelines. In order to remove primer-dimers from the PSMA PCR product and negative control, the bands of the PCR product and negative control were cut from a 0.8% agarose gel. The GENECLEAN® II Kit (Qbiogene) was used to purify the samples per manufacturer's instructions. Five microliters of the isolation products were electrophoresed on a 1.5% agarose gel to confirm the purified PCR and lack of primer-dimers.
Mouse mitochondrial DNA, PSMA transgene, and MLV-Gag PCR
Primer sequences used in these experiments.
Mouse mito F
Mouse mito R
MLV Gag F
MLV Gag R
Mitochondrial PCR: 60 ng of mouse DNA was amplified in a 25 μL reaction containing 12.5 μL 2× Gene Expression Master-Mix (Applied Biosystems), 0.5 μM Mouse mito F primer, 0.5 μM Mouse mito R primer, and 9.5 μL of water. Two microliters of 30 ng/μL mouse DNA was added the reaction. The following cycle conditions were used: 50°C for 2 minutes (UNG incubation), 94°C for 10 minutes (AmpliTaq Gold Enzyme Activation), and 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. The reactions were carried out in an Eppendorf Mastercycler personal thermocycler. The expected size of the PCR product is ~230 bp. PCR reactions were run on a 1.5% agarose/TBE gel.
Gag PCR: Varying amounts of plasmid DNA was amplified in a 25 μL reaction containing 2.5 μL 10× Titanium Taq PCR Buffer (Clonetech), 0.2 uL Titanium Taq (Clonetech), 0.2 mM dNTPs, 0.5 μM MLV Gag F primer, 0.5 μM MLV Gag R primer, and 19.8 μL of water. The following cycle conditions were used: 94°C for 1 minutes, 40 cycles of [95°C for 30 seconds and 68°C for 30 seconds], 68°C for 3 minutes. The reactions were carried out in an Eppendorf Mastercycler personal thermocycler. The expected size of the PCR product is ~500 bp. PCR reactions were run on a 1.4% agarose/TBE gel.
PSMA transgene PCR: The PSMA PCR contains 2 sets of primers to amplify two unrelated targets from PSMA transgenic mice (the transgene and genomic control sequence). Sixty to eighty nanograms of transgenic mouse DNA was amplified in a 25 μL reaction containing 12.5 μL 2× Gene Expression Master-Mix (Applied Biosystems), 1 μM S49, 1 μM INTA, 1 μM S1368, 1 μM AS2015, 6.5 μL of water, and 2 μL 30 ng/μl to 40 ng/μL mouse DNA. The following cycle conditions were used: 50°C for 2 minutes (UNG incubation), 94°C for 10 minutes (AmpliTaq Gold Enzyme Activation), 35 cycles of 94°C for 30 seconds, 58°C for 30 seconds, and 72°C for 45 seconds. The expected product sizes are ~600 bp and ~250 bp and were electrophoresed on a 1.5% agarose/TBE gel.
The PSMA PCR was also amplified with non-UNG containing taq. Sixty nanograms of transgenic mouse DNA was amplified in 25 μL reaction containing 0.5 U Taq DNA Polymerase, 10× ThermoPol Reaction Buffer (New England BioLabs, Inc.), 0.4 mM dNTP (dATP, dTTP, dCTP, and dGTP), 1 μM S49, 1 μM INTA, 1 μM S1368, and 1 μM AS2015. The following cycle conditions were used: 94°C for 5 minutes, 35 cycles of 94°C for 30 seconds, 58°C for 30 seconds, and 72°C for 45 seconds.
Negative PCR products (only water added to the PCR reaction) of the mouse mitochondrial PCR, PSMA PCR, and Gag PCR were also diluted 10-2 through 10-11 and used to simulate contamination of subsequent PCR reactions. PCR products ranged from 4 ng/μl (PSMA PCR lower band) to 10 ng/μl for all other reactions, prior to dilution for use in simulated contamination experiments.
Acknowledgements and Funding
This study was funded by NIHR01CA138444 (DOK and DJB). KMS is supported by DOD Award PC101949. The U.S. Army Medical Research Acquisition Activity, 820 Chandler Street, Fort Detrick MD 21702-5014 is the awarding and administering acquisition office. The content of this publication does not necessarily reflect the position or the policy of the Government, and no official endorsement should be inferred. AAA is the recipient of a T32 Postdoctoral fellowship (NIHT32DK007774).
- Longo MC, Berninger MS, Hartley JL: Use of uracil DNA glycosylase to control carry-over contamination in polymerase chain reactions. Gene. 1990, 93: 125-128. 10.1016/0378-1119(90)90145-H.PubMedView ArticleGoogle Scholar
- Gray ER, Garson JA, Breuer J, Edwards S, Kellam P, Pillay D, Towers GJ: No Evidence of XMRV or Related Retroviruses in a London HIV-1-Positive Patient Cohort. PLoS One. 2011, 6: e18096-10.1371/journal.pone.0018096.PubMedPubMed CentralView ArticleGoogle Scholar
- Kunstman KJ, Bhattacharya T, Flaherty J, Phair JP, Wolinsky SM: Absence of xenotropic murine leukemia virus-related virus in blood cells of men at risk for and infected with HIV. Aids. 2010, 24: 1784-1785. 10.1097/QAD.0b013e32833b76fb.PubMedView ArticleGoogle Scholar
- Oakes B, Tai AK, Cingoz O, Henefield MH, Levine S, Coffin JM, Huber BT: Contamination of human DNA samples with mouse DNA can lead to false detection of XMRV-like sequences. Retrovirology. 2010, 7: 109-10.1186/1742-4690-7-109.PubMedPubMed CentralView ArticleGoogle Scholar
- Schlaberg R, Choe DJ, Brown KR, Thaker HM, Singh IR: XMRV is present in malignant prostatic epithelium and is associated with prostate cancer, especially high-grade tumors. Proc Natl Acad Sci USA. 2009, 106: 16351-16356. 10.1073/pnas.0906922106.PubMedPubMed CentralView ArticleGoogle Scholar
- Shin CH, Bateman L, Schlaberg R, Bunker AM, Leonard CJ, Hughen RW, Light AR, Light KC, Singh IR: Absence of XMRV and other MLV-related viruses in patients with Chronic Fatigue Syndrome. J Virol. 2011Google Scholar
- Tang S, Zhao J, Viswanath R, Nyambi PN, Redd AD, Dastyar A, Spacek LA, Quinn TC, Wang X, Wood O, et al: Absence of detectable xenotropic murine leukemia virus-related virus in plasma or peripheral blood mononuclear cells of human immunodeficiency virus Type 1-infected blood donors or individuals in Africa. Transfusion. 2010, 51: 463-468.PubMedPubMed CentralView ArticleGoogle Scholar
- van Kuppeveld FJ, de Jong AS, Lanke KH, Verhaegh GW, Melchers WJ, Swanink CM, Bleijenberg G, Netea MG, Galama JM, van der Meer JW: Prevalence of xenotropic murine leukaemia virus-related virus in patients with chronic fatigue syndrome in the Netherlands: retrospective analysis of samples from an established cohort. Bmj. 2010, 340: c1018-10.1136/bmj.c1018.PubMedPubMed CentralView ArticleGoogle Scholar
- Verhaegh GW, de Jong AS, Smit FP, Jannink SA, Melchers WJ, Schalken JA: Prevalence of human xenotropic murine leukemia virus-related gammaretrovirus (XMRV) in Dutch prostate cancer patients. Prostate. 2011, 71: 415-420. 10.1002/pros.21255.PubMedView ArticleGoogle Scholar
- Aloia AL, Sfanos KS, Isaacs WB, Zheng Q, Maldarelli F, De Marzo AM, Rein A: XMRV: a new virus in prostate cancer?. Cancer Res. 2010, 70: 10028-10033. 10.1158/0008-5472.CAN-10-2837.PubMedPubMed CentralView ArticleGoogle Scholar
- Erlwein O, Robinson MJ, Kaye S, Wills G, Izui S, Wessely S, Weber J, Cleare A, Collier D, McClure MO: Investigation into the presence of and serological response to XMRV in CFS patients. PLoS One. 2011, 6: e17592-10.1371/journal.pone.0017592.PubMedPubMed CentralView ArticleGoogle Scholar
- Furuta RA, Miyazawa T, Sugiyama T, Kuratsune H, Ikeda Y, Sato E, Misawa N, Nakatomi Y, Sakuma R, Yasui K, et al: No association of xenotropic murine leukemia virus-related virus with prostate cancer or chronic fatigue syndrome in Japan. Retrovirology. 2011, 8: 20-10.1186/1742-4690-8-20.PubMedPubMed CentralView ArticleGoogle Scholar
- Hong P, Li J, Li Y: Failure to detect Xenotropic murine leukaemia virus-related virus in Chinese patients with chronic fatigue syndrome. Virol J. 2010, 7: 224-10.1186/1743-422X-7-224.PubMedPubMed CentralView ArticleGoogle Scholar
- Sakuma T, Hue S, Squillace KA, Tonne JM, Blackburn PR, Ohmine S, Thatava T, Towers GJ, Ikeda Y: No evidence of XMRV in prostate cancer cohorts in the Midwestern United States. Retrovirology. 2011, 8: 23-10.1186/1742-4690-8-23.PubMedPubMed CentralView ArticleGoogle Scholar
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