PCR and qPCR
Support and Educational Content

How to avoid false positives in PCR and what to do if you get them

General suggestions for avoiding false positives in your negative template control (NTC) sample:

  • Use freshly purified/sterile water, tubes, and reagents.
  • We highly recommend that you aliquot your probe and primers into the volumes required for a single experiment. This will decrease the chances of contamination, allow you to start with a fresh tube if you do have contamination, and mini­mize freeze–thaw cycles that can reduce oligonucleotide quality. Always include appropriate positive and negative controls in your experiment.
  • Use separate dedicated PCR work areas for reaction setup, adding the template, and handling the amplification product. Decon­taminate PCR work areas regularly with 10% bleach and UV irradiation.
  • Use 5% bleach to clean micropipettes reg­ularly. Use sterile, filter tips for pipetting to minimize contamination from aerosols. We recommend having separate pipettes and pipette tips for PCR setup and post-PCR analysis.
  • Place the no template control (NTC) wells as far as possible from positive samples.
  • Late amplification (beyond cycle 34 for SYBR® Green dye–based assays) may not be indicative of a positive NTC as it could also be a result of dimer amplification. Perform melt curve analysis after PCR to check for primer-dimers.

If you do find contamination in your NTC sample:

  • Be sure to replace all reagents and stock buffers and thoroughly clean PCR prepara­tion areas.
  • Check whether the probe is degraded. In such situations, there may still be signal from free dye and/or high background. Signal to noise assessment, mass spectrom­etry, or a fluorometric scan can be used to address possible probe degradation.
  • Use a more conserved or novel gene as a control if you are repeatedly seeing false positives.

Avoiding false positives when using universal primers for bacterial identification

In PCR experiments, amplification in the “no template control” (NTC) before the ~38th cycle with probe-based assays (or ~34th cycle when using intercalating dyes) is a sign of false positives and/or contamination. This sidebar specifically addresses false positives that occur during bacterial research when primers and probes are designed to detect common sequences, such as ribosomal RNA (rRNA).

Am I amplifying DNA from my reagents or consumables?

The exponential PCR process can amplify a single copy of DNA to detectable levels. Thus, it is important to consider the pervasiveness of the chosen primer or probe sequences. There are some DNA sequences, such as bacterial genes for 16S or 23S rRNA, that can be found almost anywhere. While not commonly used for RT-PCR or gene expression studies, rRNA sequences are often used for characterizing environmen­tal species diversity, such as bacterial strains within the intestine or in salt water marshes. For such applications, genomic DNA, rather than RNA or cDNA, is used as the sample.

Choose a well-conserved, species-specific gene or novel sequence

In 16S rRNA experiments, a better approach may be to choose a unique sequence from the hypervariable region of 16S rRNA. Alternatively, a conserved, species-specific gene may be used. Blocking oligos and/or clamps can also be used to block amplification of common sequences and enhance the amplification of a rare sequence.

As new sequences are deposited into the NCBI database, it is essential to perform a BLAST search of every primer and probe sequence used in PCR to check for specificity and cross reactivity.

Bacterial ribosomal sequences can be amplified readily from virtually any bacterial source, including bacteria-derived Taq polymerases used to amplify them, as well as nonsterile tubes and pipette tips. Hence, if a positive NTC is observed, testing with different master mixes will help rule out the master mix as the cause of contamination.

Product focus: Reagents for PCR

All the reagents you need for successful qPCR assays are available through IDT.

Additional reading

Sample preparation for successful qPCR—Read about qPCR sample preparation, and what experimental details you should consider for obtaining accurate and consistent results.

Steps for a successful qPCR experiment—Read these recommendations for 5′ nuclease assay design and experimental setup that will help you obtain accurate and consistent results.

Design efficient PCR and qPCR primers and probes using online tools—Simplify planning of your qPCR experiments using IDT free, online tools for oligonucleotide analysis and PCR primer design. This article provides an overview of our predesigned qPCR assays and the basics of designing customized PCR primers and hydrolysis probes with the PrimerQuest® Tool.

Review other DECODED Online newsletter articles on PCR and qPCR applications.

You can also browse our DECODED Online newsletter for additional application reviews, lab tips, and citation summaries to facilitate your research.

Author: Darcey Klaahsen, MS, is a Scientific Applications Specialist at IDT.

© 2013, 2016 Integrated DNA Technologies. All rights reserved. Trademarks contained herein are the property of Integrated DNA Technologies, Inc. or their respective owners. For specific trademark and licensing information, see www.idtdna.com/trademarks.

Predesigned qPCR Assays

Probe-based qPCR assays for quantification of human, mouse, and rat gene expression. Order in plates or tubes.

Search human, mouse, or rat genes ≫

Related Articles

Designing PCR Primers and Probes

General guidelines for designing primers and probes and for choosing target locations for PCR amplification.

Read more ≫

Steps for a Successful qPCR Experiment

Considerations for 5′ nuclease assay design and experimental setup to help you obtain accurate and consistent results.

Read more ≫

Interpreting Melt Curves: An Indicator, Not a Diagnosis

Examining PCR melt curve data to determine what it can/cannot tell us about resulting PCR amplicons.

Read more ≫

Epigenetic Biomarkers for Prostate Cancer

Scientists use methylation and expression analysis methods to evaluate epigenetic markers for early, noninvasive detection of aggressive prostate cancer. IDT PrimeTime® qPCR Assays, ZEN™ Double-Quenched Probes, and gBlocks® Gene Fragments facilitate this research.

Read more ≫

Optimizing Multiplex qPCR for Detecting Infectious Diseases and Biothreat Agents in the Field—ZEN™ Double-Quenched Probes bring down the background

Tetracore researchers developing large sets of robust probe-based qPCR assays discuss the need to: use probe dyes compatible on common PCR instruments, maintain low background with multiple probes, and reformulate assays to address viral mutation.

Read more ≫