Sample adequacy controls (SAC) for molecular detection methods

James Woodhall,¹ Lina Rodriguez Salamanca,² and Telissa Wilson^3
1University of Idaho, Parma, ²Virginia Tech, ³Washington State Department of Agriculture
 
Several controls are often recommended for qPCR and other molecular detection methods, such as LAMP.  There is a wide range of terms and approaches people can use. Most people use a positive and negative (blank) control, but perhaps the Sample Adequacy Control (SAC) should be used more in plant molecular diagnostics. The SAC is another PCR targeting a DNA sequence guaranteed to be in the sample. It has been described as a “built-in check” (Brukner et al., 2021). For example, if testing for a pathogen in potato tubers, you would use an assay that detects potato DNA. The SAC has advantages over other methods, as it is the true test that nucleic acid has been successfully extracted and the resulting DNA is suitable for amplification (PCRable). Positive amplification of the SAC verifies that no conflicting inhibitors are present in the nucleic acid extract and that the sample DNA is of sufficient quality and quantity to be detected. They are an effective check for false negatives.
 
At the University of Idaho’s Parma lab, two assays are routinely used as quantitative SACs to ensure the nucleic acid extraction has been successful and to minimize false negatives. These are the qPCR assay using the COX loci for plant material (Tomlinson et al., 2005) and the universal 16s bacterial assay (Yang et al., 2002). The universal bacteria assay is used with DNA extracted from soil to ensure the extraction has been successful and inhibitors are not present; generally, any soil DNA sample generating a Ct value over 26 with this assay is considered to not have sufficient quality to trust the result of the pathogen-specific PCR assays. The universal bacteria assay is also used in troubleshooting and method development of nucleic acid extraction methods, as is the FungiQuant assay (Liu et al., 2012). The assays described are listed in Table 1, along with a plant COX assay which can be used as the SAC for LAMP reactions.
 
Typically, these assays are run as a separate reaction as there are often concerns about decreasing the pathogen-specific assay’s sensitivity through competitive inhibition when used as an internal control in a multiplex reaction. This inhibition (i.e., an increase in Cq value) may not be a problem for some high-titer pathogens, which is why you sometimes see papers describing assays multiplexed with internal controls published. However, the increase in Cq can pose problems when detecting pathogens in soil or in asymptomatic plant material where low-level detection is key. Therefore, one issue with SACs is that it does increase the cost as additional reactions are often required. Another drawback is the SAC Cq values may not be consistent between different types of samples. For example, when using the real-time PCR COX assays in Table 1, the Cq values generated from wheat plants are significantly higher than those generated for potatoes, even if the extracted DNA quantity is the same. There is a wide range in Cq values generated for soil with the universal bacteria assay simply because soil is one of the most diverse substances known.
 
One way the Cq variance of the internal control can be mitigated is by using an exogenous positive control (DNA or even an organism known to be unrelated to the target spiked into the sample at a known level). Several companies even offer synthetic DNA sequences, which can be used as exogenous controls. However, the exogenous control does change the sample, which may impact other methods that can later be used to analyze it, such as high throughput sequencing. Also, spiking in samples can impact the judgment of any potential sample deterioration, plus it may be harder to judge PCRability, particularly if the spiked DNA is at a high level. When using synthetic exogenous controls, some diagnosticians combat this issue by spiking the control into the sample pre-extraction.
 
The benefits of SACs are discussed further in Brukner et al. (2021), who state that they can have an important analytical, clinical, and epidemiological value and can increase confidence in a negative test result. At the Parma lab, they are found to be an invaluable tool in the screening services offered, method development and assay troubleshooting. When considering a molecular detection assay that does not include an internal control target, we recommend that the costs and benefits of including a SAC be considered.
 
Table 1. Primer and probe sequences of assays used as Sample Adequacy Controls for plant diagnostics at University of Idaho, Parma.
 

 *FAM-BHQ1 probes are used at University of Idaho, Parma.
 
Brukner, I., Resendes, A., Eintracht, S., Papadakis, A. I., and Oughton, M. 2021. Sample Adequacy Control (SAC) Lowers False Negatives and Increases the Quality of Screening: Introduction of “Non-Competitive” SAC for qPCR Assays. Diagnostics 11,1133. https://doi.org/10.3390/diagnostics11071133
 
Liu, C.M., Kachur, S., Dwan, M.G., Abraham, A.G., Aziz, M., Hsueh, P-R., Huang, Y-T., Busch, J.D., Lamit, L.J., Gehring, C.A., Keim, P., and Price, L.B. 2012. FungiQuant: A broad-coverage fungal quantitative real-time PCR assay. BMC Microbiology 12, 255. https://bmcmicrobiol.biomedcentral.com/articles/10.1186/1471-2180-12-255
 
Tomlinson, J.A., Boonham, N., Hughes, K.J.D., Griffin, R.L., and Barker, I. 2005. On-site DNA extraction and real-time PCR for detection of Phytophthora ramorum in the field. Applied and Environmental Microbiology 71, 6702–6710.  https://journals.asm.org/doi/epub/10.1128/AEM.71.11.6702-6710.2005
 
Tomlinson, J.A., Dickinson, M.J., and Boonham, N. 2010. Rapid detection of Phytophthora ramorum and P.  kernoviae by two-minute DNA extraction followed by isothermal amplification and amplicon detection by generic lateral flow device. Phytopathology 100, 143-149. https://apsjournals.apsnet.org/doi/pdf/10.1094/PHYTO-100-2-0143
 
Weller, S.A., Elphinstone, J.G., Smith, N.C., Boonham, N., and Stead, D.E. 2000. Detection of Ralstonia solanacearum strains with a quantitative, multiplex, real-time, fluorogenic PCR (TaqMan) assay. Applied and Environmental Microbiology 66, 2853-2858. https://journals.asm.org/doi/epub/10.1128/AEM.66.7.2853-2858.2000
 
Yang, S., Lin, S., Kelen, G.D., Quinn, T.C., Dick, J.D., Gaydos, C.A., and Rothman, R.E. 2002. Quantitative multiprobe PCR assay for simultaneous detection and identification to species level of bacterial pathogens. Journal of Clinical Microbiology 40, 3449–3454. https://journals.asm.org/doi/10.1128/JCM.40.9.3449-3454.2002
 
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