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  • Writer's picturePaul Campbell

With qPCR, primer selection is everything!




qPCR is an incredibly powerful tool for tracking keystone microbial groups in wastewater treatment plants (WWTPs) - but it's a tool with some idiosyncrasies. The biggest? You get what you test for. The primers used in a qPCR reaction determine what target template(s) will be amplified. If you choose the wrong primer set for the sample being tested, the results may be very misleading. Case in point? Tracking nitrification using the most common qPCR assay for the amoA gene, which is based on a 1997 paper by Rotthauwe and colleagues. The researchers indicate that the primer set will detect the amoA gene for Nitrosomonas europaea and Nitrosomonas eutropha along with several strains of Nitrosospira (an AOB, not to be confused with Nitrospira!!). This primer pair has since been used in a considerable number of peer-reviewed journal articles without questioning if the primer set will reflect the actual organisms working in the wastewater treatment systems.


First, what are primers? Primers are small, synthetic pieces of DNA (oligonucleotides) that are synthesized in a lab. The sequence of the primer (the sequence of G, A, T and C that make up the primer) complement the target to be amplified and provide a starting point for DNA synthesis. The wikipedia article does a great job of explaining primers and the polymerase chain reaction.


We routinely use qPCR to monitor the nitrifier populations of different WWTPs. In our experience, Nitrosomonas are the dominant ammonia-oxidizing bacteria (AOB) present in most systems, while Nitrospira are the dominant nitrite-oxidizing bacteria (NOB). (Of course, there are exceptions to the rule.) But, each WWTP has a different influent composition which, in turn, influences which nitrifiers will grow in that particular system. Assuming that Nitrosomonas europaea and Nitrosomonas eutropha are always the dominant AOBs can lead to some real misunderstandings.


To demonstrate this, we analyzed different samples using different qPCR primer sets. We used the amoA 1F/2R primer pair from the paper cited above, along with 2 additional sets of primers designed in-house (our custom primer sets detect over 10 known species). All four samples were known to come from nitrifiying systems, verified through microbial community analysis (MCA). The results are in the table below:

Site

amoA 1F/2R

N. marina

N. nitrosa

% by MCA

Refinery 1

1.3%

2.1%

1.4%

2.1%

Refinery 2

0.1%

9.8%

0.1%

1.8%

Chemical Plant

2.8%

0.0%

9.9%

3.0%

Food Processing

0.0%

0.0%

0.0%

2.3%

The original amoA 1F/2R primer pair detected the Nitrosomonas present in the samples from Refinery 1 and the Chemical Plant, but failed to pick up the other two. And, while the primer sets designed against N. marina and N. nitrosa detected the AOBs in Refinery 1, these custom primers worked very differently for Refinery 2 and the Chemical Plant. Finally, none of the 3 tested primer sets worked on the Food Processing sample!


Bottom line? It really helps to use the correct primer set when monitoring nitrification in WWTPs. Our advice is to:


  • Collect a baseline sample when nitrification is working well in your facility and use MCA to determine which AOB and NOB are present in your system

  • Test multiple primer sets to find the one(s) that work best for your facility

  • Occasionally re-test fresh samples with multiple primer sets as the nitrifier population can shift over time


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