qPCR for phage therapy, part II

Issue 218 | April 7, 2023
16 min read
Capsid and Tail

This week Jessica Sacher continues her ‘qPCR for phage therapy’ series. She takes us through how we know if our qPCR worked, what to look for, and how to interpret how much phage/bacteria were in our samples based on the results.

Check out part I here if you missed it!

What’s New

Phages seem to be saving lives left and right, but still, they remain unapproved drugs in most countries. Pharma companies don’t sell them; hospitals and pharmacies don’t stock them. In this Wired article, Maryn McKenna covers phage therapy; why we don’t have it yet, and where it’s at. A great one to share with family and friends!

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MagazinePhage therapyScience communication

In a new paper in Nature, Joseph Kreitz (Howard Hughes Medical Institute) and colleagues show that Photorhabdus virulence cassette (PVCs), part of a class of syringe-like nanomachines resembling phage tails, can be reprogrammed for protein delivery in eukaryotic cells.

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Drug deliveryNanomachinesResearch paper

By analyzing metagenomic samples from IBD patients, Shaqed Carasso (Technion-Israel Institute of Technology) and colleagues identified invertible regions that correlate with disease, including Bacteroides fragilis polysaccharide-A, which is mostly ‘OFF’-switched during inflammation, but switches ‘ON’ when inflammation is resolved. Intriguingly, they found that phages seem to induce the ‘OFF’ switch…

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Research paperInflammatory Bowel Disease

This week’s EMDB (Electron Microscopy Data Bank) release includes this beautiful 4.4 Å single particle CryoEM structure of a phicrAss001 virion!

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CrAssphageEMDBStructural biology

This review by Fazal Mehmood Khan (Shenzhen University) and colleagues provides an update on the use of animal models in phage therapy, summarizing preclinical approaches in various in-vivo models and highlighting the need for pharmacokinetic data before application in humans.

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Animal modelsPhage therapy

Latest Jobs

Sponsored Ad Phages in agricultureMicrobiologist
Research Scientist at A&P Inphatec in Palo Alto, CA

A&P Inphatec, LLC has commercialized the first product to treat Pierce’s Disease in grapevines. Our unique solution utilizes bacteriophages to target and kill the bacteria at the source of the problem – giving wine growers an organic and sustainable solution to a rising threat in California.
A&P Inphatec, LLC is seeking an experienced microbiologist applicant to join our research and development team as a full-time Research Scientist.

MicrobiologyPhage characterizationMSc Project
The Bhabha Atomic Research Centre Facilities (India) are seeking an MSc final-year student for a 6-month project on the isolation and characterization of phages from environmental samples. The position is open only for Indian nationals.
FellowshipPost BacPost Doc
The US FDA is seeking applications for three phage therapy-related research opportunities (open to post-baccalaureate, post-masters, and postdocs). Projects are aimed at evaluating the use of phage to treat Staphyloccocus aureus colonization and/or disease in order to better combat AMR pathogens.
MicrobiotaWomen's healthPhage therapyPrincipal Researcher
The Department of Microbiology, Tumor and Cell Biology at Karolinska Institutet is seeking a Principal Researcher to focus on phage therapy and microbiota in women’s health.
Animal modelsPhage therapyResearch assistant
The Lee Kong Chian School of Medicine (Singapore) is seeking a Research Assistant to work with Dr. Kevin Pethe on evaluating therapeutic phages in animal models of bacterial infections.

Community Board

Anyone can post a message to the phage community — and it could be anything from collaboration requests, post-doc searches, sequencing help — just ask!

Hello Phage People,
Does anyone use Phage DNA Isolation Kit from Norgen Biotek? I’m using this kit and trying to increase the DNA concentration. I already did all the additional steps but does anyone have any tricks for increasing concentration? I appreciate it if you mail me your tricks.
Best, Irem

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Phage methodsSeeking suggestions

The recording of the recent Clubhouse audio conversation on GMP phage therapy production (’Just say no to GMP?’) has just been posted! This was a talk hosted last week by Sabrina Green, Adriana Hernandez, Jean-Paul Pirnay, Patrick Druggan, and Barbara Brenner.

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Phage manufacturingPhage therapyVirtual Event

The UK parliament recently heard evidence on phages for AMR. According to Stephanie Lesage via LinkedIn: “There was a general consensus on the need to embrace the biological and unique nature of phages and find more suitable approaches to stringent GMP production, but also make regulations more flexible by adopting a more pragmatic framework to ensure phages/cocktails can be updated to follow epidemiological evolutions and the unique requirements of some patients.”

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ParliamentPhage therapy

Viruses of Microbes is happening this year in Tbilisi, Georgia July 3rd-7th. The deadline for abstract submission is May 1st.

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ConferenceViruses of Microbes

The Canadian Society of Microbiologists conference (CSM) is hosting a phage session (Bacteriophage and bacterial defense systems). This year, the conference is hosted in Halifax from June 25th-28th, and the abstract deadline is April 17th.

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ConferencePhage defense systems

The International Workshop on Ecophage is happening in Valencia, Spain from Sept 12-13, 2023. Join experts in phage research, farmers, food companies, policy makers, and consumer reps in a workshop on phage-based strategies for preventing AMR in the agri-food system.

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Top Agar Question

This is a basic question but one I do need advice with. My high school phage team will begin looking for a phage next fall from a new host. The host has not been studied much for phages. How do you choose a top agar for new hosts/phages? Is there a generic TA that works well? We currently use MBTA for Mycobacterium but I did not know if top agars are phage specific. Thanks for your guidance.

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Lab tipsPhage microbiology

qPCR for phage therapy, part II

Profile Image
Phage microbiologist and co-founder of Phage Directory
Co-founderPostdoctoral Researcher
Iredell Lab, Phage Directory, The Westmead Institute for Medical Research, Sydney, Australia, Phage Australia

Phage characterization, Phage-host interactions, Phage Therapy, Molecular Biology

I’m a co-founder of Phage Directory and have a Ph.D in Microbiology and Biotechnology from the University of Alberta (I studied Campylobacter phage biology). For Phage Directory, I oversee community building, phage sourcing, communications, science, and our awesome team of volunteers.

As of Feb 2022, I’ve recently joined Jon Iredell’s group in Sydney, Australia as a postdoctoral research scientist for the Phage Australia project. I’m diving back into the lab to help get Phage Australia’s country-wide phage therapy system up and running here, working to streamline workflows for phage sourcing, biobanking and collection of phage/bacteria/patient matching and monitoring data, and integrating it all with Phage Directory’s phage exchange, phage alerts and phage atlas systems. I’m also delving into phage manufacturing and quality control.

Welcome to part II of the ‘qPCR for phage therapy’ series!

In part I, I introduced qPCR as an underutilized way to track phages and bacteria during phage therapy. I went over the basic steps and why we do them, why we need qPCR standards and how to make them, and how I’m thinking about it all as I start to run qPCR on patient serum samples.

For part II, I wanted to go into what we should be looking for as we analyze our results. Did our primers work? Was our PCR reaction efficient? Is our standard curve useable? Assuming all these answers are yes, it’s finally time to take a look at our samples: how much phage / bacteria do we see in our patient serum samples?

Read part I here first if you missed it: qPCR as a window into phage therapy

⛔ Reminder: I am new to qPCR! My goal is to give a high-level overview of what I’m learning as I dive into this new technique. There are way more detailed/complete guides online if you’re ready to run qPCR yourself! Please only read the below as an intro/overview, and not as a complete tutorial.

Did our qPCR assay work?

So your qPCR run is complete, and you’ve got the data. So exciting! How many phages (or bacteria) were in your samples?

Not so fast. I like to hold off on checking my samples until I’ve had a good look at my standard curves, controls, and melt curves. If these don’t look right, we can’t interpret much about our samples anyway. A few things to look for:

Check PCR efficiency and limit of detection

You’ll want to look for several things. First, did your primers work? To get a sense, you’ll want to check out your standard curve (most likely, the software will plot it for you). Does it look nicely linear or not? As a reminder, the standard curve is made by graphing the Cq value for all of your standards (which, as we discussed in part I, would have come from decreasing, known concentrations of your starting phage or bacteria — whatever you’re trying to quantify).

When the Cq of your standards is graphed against the ‘starting quantity’ (ie. the known amount in PFU/mL that you added to your standards, on a log scale), the line should look linear, with a good (ie. close to 99%) r^2. The machine will also give you a PCR efficiency readout; you want 90-110% (anything lower or higher means your primers aren’t working efficiently).

Your replicates should be clustering close together for most standards; though maybe some of the replicates start to diverge from each other at very low DNA concentrations (high Cq). This is to be expected. The point at which the relationship isn’t linear anymore is the point we can’t rely on our standard curve, and thus can’t use it to convert our sample Cq values into PFU/mL numbers.

fig 1 std curve w LOD pic
Fig 1: Example of a standard curve. It looks linear when you graph the log of PFU/mL vs. Cq (except at very high Cqs; the replicates start spreading more, the linear relationship breaks down; the curve becomes less reliable). The red dotted line shows where we might place the limit of detection, ie. the reliability limit for our assay. (In this example, it looks like readings under 10^2 PFU/mL are too unreliable, so we’d call that our limit of detection)

Check if your PCR was specific

You can also check the melt curve for each reaction, which helps tell you about quality of your reactions. Check for a nice, single peak (what you want), or for two peaks, or a peak with a shoulder (what you don’t want; suggests multiple products, likely primer dimers or nonspecific amplification).

fig 2 melt curve pic
Fig 2: Black shows a single peak (good, this means a single product); red shows a shoulder (less ideal; shows there’s some non specific amplification, maybe primer dimers).

If any samples or standards show ugly melt curves, it could be a sign your PCR reaction isn’t working quite right. It could be the primers — maybe they need to be redesigned? It could be a bunch of other things too (there are many troubleshooting guides online).

Check that your negative controls are negative

Check your controls: are you getting amplification in your no-template control (NTC)? If so, something in your master mix (maybe the water you used?) might have a DNA contaminant. Any amplification in your crossover control? If so, you have one set of primers amplifying the wrong phage… you don’t want this, and should redesign your primers (maybe to amplify a different gene; something the other phage definitely doesn’t have, ideally).

Check that samples are within your standard curve range

Now check your samples. Are the Cq values within the range of your standards? (ie. not higher than the highest standard; not lower than the lowest).

fig 3 standard curve range
Fig 3: Your standard curve dictates the range of Cq values you can reliably convert to PFU/mL. In this illustration, Cq values between 17 and 34 are fine; anything below 17 or above 34 would be uninterpretable (you’d need to go make some additional standards). Looking on the y-axis, this means the range you can accurately measure in your samples (with these primers, for this target, under these conditions) is between 10^2 and 10^7 PFU/mL.

Samples (finally!): how much phage (or bacteria) did we count?

Assuming you filled in the ‘known quantities’ for your standards, the software you’re using (that comes with the qPCR machine) will calculate the predicted PFU/mL (or CFU/mL) that your samples have in them. Yay! These are the values you want. You can graph them and do stats on them to your heart’s content.

⛔ Caveat: qPCR detects DNA copies, not actual PFU/mL or CFU/mL; but your standards were set at a certain PFU/mL or CFU/mL (and depending on how many dead/inactive ones there were in there, this number may or may not reflect the number of copies of DNA in that organism). For simplicity, you may want to assume 1 copy = 1 PFU, and swap out the units (ie. report ‘copy number per mL’ instead of PFU/mL). This is a larger topic to explore for another day.

Now it’s finally time to think about the data and what it might mean biologically. Did your phage go up higher after an hour in the body? Did it drop quickly back to baseline after a day? There’s so much you can glean; of course depending on when you were able to get samples.

In a future post, if there’s interest, I’m happy to get into how to actually turn Cq values into PFU/mL (or CFU)/mL yourself, along with a spreadsheet tutorial, so you can understand what the software is doing, rather than being reliant on it. Let me know if that’s something you’d be interested in seeing!

Admire your handiwork

Great work! You’ve done qPCR. Do this for every organism (phage or bacteria) you want to detect in your samples. Do this over time during phage therapy, and track what’s going on in the patient’s body (obviously this needs to be part of an ethics-approved trial since it involves human subjects). Or just use it count phages/bacteria copies in any sample you come across. Whatever you want!

Of course, don’t forget that even if your qPCR is perfect, with all the right controls and standards, it is never going to give you a full picture of what’s going on biologically. It’s a method of counting DNA copies, and it doesn’t distinguish between living vs. dead phages/bacteria. That said, I still think it’s a helpful way to add colour to what’s going on with the phage/host battles we’re facilitating, from the test tube to the patient.

The beauty of a well-established method

A reminder that even though this method is rarely done in the phage therapy field, it’s super common in the world in general! This means there’s a LOT more you can learn about qPCR; lots of detailed troubleshooting and experimental design guides online. I am barely scratching the surface here, and am certainly still learning myself. But I hope this has given you a window into roughly what qPCR is about, how it might be applied to phage-related questions, and helped you get a sense of the process before you get lost in too many rabbit holes and get too overwhelmed to start.

Further reading

Capsid & Tail

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