By James Woodhall (University of Idaho, Parma), Lina Rodriguez Salamanca (Iowa State University)
PCR inhibition is a common obstacle when working with plant materials because of compounds present in soil and roots, as well as high polysaccharide levels present in some tissues. There are many strategies to cope with PCR inhibitors, and it is often best to employ several approaches to increase the robustness of your method. Below, we discuss some of the approaches we have successfully used in our labs.
Sample collection and preparation: Sample collection can be refined to reduce the collection of PCR inhibitors. Removing plant material that is high in polysaccharides and that may not contain the pathogen is one approach. Soil contains humic acid, which is one of the most potent PCR inhibitors. Thoroughly washing samples in the lab can reduce the presence of soil.
Nucleic acid extraction: Robust nucleic acid extraction methods can eliminate or reduce PCR inhibitors considerably. The various Qiagen soil DNA extraction kits contain Inhibitor Removal Technology (IRT) which eliminates humic acid content. Sometimes, a DNA separation strategy can be effective at removing inhibitors, such as using magnetic particles rather than a spin column flow-through technique. One method for extracting DNA from large quantities of soil utilizes a two-stage DNA separation process for reducing PCR inhibitors as well as concentrating the sample [1]. The first stage uses glass milk (silicon dioxide suspension), and the second stage uses paramagnetic particles from Promega.
Post nucleic acid extraction approaches: The simplest approach for this is to do a tenfold dilution of your DNA extraction. Although you risk losing sensitivity, in some cases you will improve your PCR results as you have diluted your PCR inhibitors to below the threshold for inhibition. There are various DNA and RNA cleanup kits that can remove inhibitors from your extracts. Paramagnetic beads such as AMPure XP can also be used for a post-extraction cleanup.
PCR enhancers and robust master mixes: The type of PCR used also influences the impact of inhibitors. TaqMan PCR appears to be much more tolerant of the presence of PCR inhibitors than SYBR green methods. Master mix can play a key role, too. Products from ThermoFisher such as Environmental Master Mix 2.0 and TaqMan Fast Virus 1-Step Master Mix can tolerate high levels of humic acid inhibition in real-time PCR and reverse transcriptase real-time PCR, respectively. Perfecta qPCR Tough Mix also shows a good tolerance of PCR inhibitors. Skim milk powder and bovine serum albumin added to PCR can also mitigate the effect of PCR inhibitors.
Troubleshooting
If you suspect you have a PCR inhibition issue, obtaining and comparing values from the nanodrop could be your first step at troubleshooting. PCR inhibition tests are also useful. This is when you use another PCR assay specific to something not present in your sample DNA. When you set up your PCR plate you add your sample DNA extract to a fixed amount of DNA (called exogenous DNA) that is detected by the other PCR assay. If your Ct values are higher when the exogenous DNA is in the presence of sample DNA, than by exogenous DNA alone, you know you have PCR inhibitors present. Another approach is to use an endogenous internal control; this is DNA that is present in all samples tested. Some labs spike in a plasmid, synthetic DNA (gblocks or genestrands) or an organism (such as a bacterium or yeast) that is known to be absent from the sample and have a corresponding TaqMan assay. Another approach could be using universal assays for plant material [2] or bacteria [3] and monitoring any deviations in Ct.
Do you have other approaches for coping with PCR inhibition? If so, please let us know! Email us at lina@iastate.edu
References
PCR inhibition is a common obstacle when working with plant materials because of compounds present in soil and roots, as well as high polysaccharide levels present in some tissues. There are many strategies to cope with PCR inhibitors, and it is often best to employ several approaches to increase the robustness of your method. Below, we discuss some of the approaches we have successfully used in our labs.
Sample collection and preparation: Sample collection can be refined to reduce the collection of PCR inhibitors. Removing plant material that is high in polysaccharides and that may not contain the pathogen is one approach. Soil contains humic acid, which is one of the most potent PCR inhibitors. Thoroughly washing samples in the lab can reduce the presence of soil.
Nucleic acid extraction: Robust nucleic acid extraction methods can eliminate or reduce PCR inhibitors considerably. The various Qiagen soil DNA extraction kits contain Inhibitor Removal Technology (IRT) which eliminates humic acid content. Sometimes, a DNA separation strategy can be effective at removing inhibitors, such as using magnetic particles rather than a spin column flow-through technique. One method for extracting DNA from large quantities of soil utilizes a two-stage DNA separation process for reducing PCR inhibitors as well as concentrating the sample [1]. The first stage uses glass milk (silicon dioxide suspension), and the second stage uses paramagnetic particles from Promega.
Post nucleic acid extraction approaches: The simplest approach for this is to do a tenfold dilution of your DNA extraction. Although you risk losing sensitivity, in some cases you will improve your PCR results as you have diluted your PCR inhibitors to below the threshold for inhibition. There are various DNA and RNA cleanup kits that can remove inhibitors from your extracts. Paramagnetic beads such as AMPure XP can also be used for a post-extraction cleanup.
PCR enhancers and robust master mixes: The type of PCR used also influences the impact of inhibitors. TaqMan PCR appears to be much more tolerant of the presence of PCR inhibitors than SYBR green methods. Master mix can play a key role, too. Products from ThermoFisher such as Environmental Master Mix 2.0 and TaqMan Fast Virus 1-Step Master Mix can tolerate high levels of humic acid inhibition in real-time PCR and reverse transcriptase real-time PCR, respectively. Perfecta qPCR Tough Mix also shows a good tolerance of PCR inhibitors. Skim milk powder and bovine serum albumin added to PCR can also mitigate the effect of PCR inhibitors.
Troubleshooting
If you suspect you have a PCR inhibition issue, obtaining and comparing values from the nanodrop could be your first step at troubleshooting. PCR inhibition tests are also useful. This is when you use another PCR assay specific to something not present in your sample DNA. When you set up your PCR plate you add your sample DNA extract to a fixed amount of DNA (called exogenous DNA) that is detected by the other PCR assay. If your Ct values are higher when the exogenous DNA is in the presence of sample DNA, than by exogenous DNA alone, you know you have PCR inhibitors present. Another approach is to use an endogenous internal control; this is DNA that is present in all samples tested. Some labs spike in a plasmid, synthetic DNA (gblocks or genestrands) or an organism (such as a bacterium or yeast) that is known to be absent from the sample and have a corresponding TaqMan assay. Another approach could be using universal assays for plant material [2] or bacteria [3] and monitoring any deviations in Ct.
Do you have other approaches for coping with PCR inhibition? If so, please let us know! Email us at lina@iastate.edu
References
- Woodhall, J. W., et al. A new large-scale soil DNA extraction procedure and real-time PCR assay for the detection of Sclerotium cepivorum in soil. Eur J Plant Pathol. 134, (3), 467-473 (2012).
- Tomlinson, J. A., et al. On-Site DNA Extraction and Real-Time PCR for Detection of Phytophthora ramorum in the Field. Appl Environ Microbiol. 71, (11), 6702-6710 (2005).
- Yang, S., et al. Quantitative multiprobe PCR assay for simultaneous detection and identification to species level of bacterial pathogens. Journal of Clinical Microbiology, 40 , 3449–3454 (2002).
