A team led by researchers from Sweden's Uppsala University has developed a real-time PCR assay that can broadly detect animal and human influenza viruses A, B, and C.
The assay is the first single-step, multiplex PCR method that enables simultaneous detection of a broad range of the three influenza types, according to the researchers, who describe the work in a paper that is currently in press with the Journal of Virological Methods.
One key to the assay is the use of the nucleoprotein gene as a target. While a number of flu assays rely on matrix genes, the authors note that the NP gene is more strongly conserved for influenza B and C than the matrix gene, making it a "a good choice for PCRs that cover a wide range of targets." In addition, they note, "influenza types A, B, and C are based on the antigenicity of the NP gene, and it is, therefore, logical to detect and simultaneously type the three influenza types using NP as a target."
The researchers, led by Jonas Blomberg of the Department of Medical Sciences at Uppsala University, also developed a new probe to target the highly variable influenza A target sequence. The probe, called MegaBeacon, or MegB, is a "hybrid" between a molecular beacon and a TaqMan probe because it is nuclease-degradable, like a TaqMan probe, but much longer and mismatch-tolerant, like a molecular beacon.
"The basis of the MegB strategy is to extend the length of the probe to increase its [melting temperature] and to disperse hybridization over a longer nucleotide stretch, encompassing variation in the entire region," Blomberg and colleagues wrote. "As long as there are sufficient perfectly matching sequence regions, the probe will bind."
The researchers add that the increased fault tolerance of MegB "has the potential to improve detection of variable viruses in general and to enable detection of entire virus families, such as Coronaviridae."
PCR Insider spoke to Blomberg recently about the MegaBeacon strategy and its potential uses. An edited version of the interview follows.
Can you provide a bit of background for this work? Is this the first assay developed to detect influenza A, B, and C?
Yes, I think it's the only one. This is not entirely complete, but very broadly covers orthomyxaviruses with PCR. It's the first time that this new principle, the MegaBeacon, is introduced into the literature. It's very flexible. With this design, we can achieve new functionality of TaqMan probes.
The TaqMan probes are relatively short — generally below 30 nucleotides — and you can't make longer ones because then you must introduce a quencher inside the probe and that is difficult from a synthetic point of view. So we wanted to be able to stretch out the probe over a much longer target than was possible with ordinary TaqMan probes.
We got this idea that if we make a hairpin out of the TaqMan probe, this way the quencher is next to the fluorophore — just like a molecular beacon. But this is much more than a molecular beacon. It's also five-prime nuclease degradable. So it's a combination of TaqMan and a molecular beacon.
In this way, we can have the advantages of both. So there is no limit — not a very big limit, anyway — on how big it can be. I think you can have 70 or 80 or so nucleotides as a probe. This gives the possibility of evening out mismatches. So the one we have in this paper can tolerate up to 10 mismatches, but with longer probes, and of course depending on the target variability, we probably could have even higher tolerance. And for an ordinary TaqMan probe, it can only handle a few mismatches and then it just dies.
So it's a very useful strategy if you have variable targets like RNA viruses — the orthomyxaviruses being prime examples, and also for retroviruses. We have designed a [xenotropic murine-like retrovirus] system that detects the XMRV envelope gene, which is highly variable — that's also with MegaBeacon. And we have done a coronavirus system, but those are not published yet.
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So the reason that no one has been able to do this in the past was because they didn't have a probe long enough to cover all the variation across all three viruses?
There are systems that use the matrix gene, which is one of the more conserved in the influenza viruses. Those are broadly targeted also, but we wanted to use the nucleoprotein gene, which is relatively conserved, because it's the basis for division between influenza A, B, and C. So it's logical. If you want to say something is influenza B, you do that because of its properties in the nucleoprotein. So it's a logical step if you want to characterize something.
So that's one thing. The other is that it has not been possible to use the more variable targets before this new technique.
The paper notes that the MegaBeacon probe was really only required to detect influenza A. Can you discuss why that is the case?
That is because influenza A is more variable than influenza B and C. So we did construct a normal TaqMan for influenza A before, but we found that we lost some strains, and it was a bit frustrating just to chase point mutations. We wanted something much more mismatch-tolerant. And then this idea with the MegaBeacon came up.
But we have found it working fine in coronaviruses, where we can detect the whole coronavirus family with this MegaBeacon probe. And also XMRV.
Are there any disadvantages to this method, especially as it extends to 70 or 80 nucleotides?
Well, there is a tendency for some stretches to have a secondary structure, and it can be a little hard to predict, so I suggest that one does careful designing in various programs like M-fold or Visual OMP and so on. You must do your informatics in order to avoid unpleasant surprises.
How are you using this assay in practice? Are other labs adopting it?
The XMRV [assay] is brand new and that is only used in my own lab. The coronavirus [assay] has spread to another lab. And the influenza [triplex assay] has also spread to another lab. I've encountered great interest in colleagues for this.
What would be advantages of doing all this with one test instead of separate tests? Would it be primarily cost savings or time savings?
Often the influenzas are not properly diagnosed. There are rapid tests that are antigen-based immunoassays, and they are, first, very expensive, and they also have a low sensitivity. So there is really a clinical need for broadly detecting influenza with PCR.
To some degree you can do this with matrix-gene based PCRs, but influenza C is highly neglected. Because no one tests for it, people think that it's not present, so it's a very anonymous virus. So by including it, we can find it in, for example, children. Nowadays, the level of ambition — especially for respiratory virus detection — has been raised, so people expect to see the whole picture, including such things as rhinoviruses. So this is a step in that direction.
We're also working on multiplex systems using the Luminex system. So we also have further developments beyond this MegaBeacon method. We actually have a patent on a system to detect many things at the same time using Luminex.
Has that work been published yet?
No, but the patent has been submitted, so we're talking about it in meetings. It's going to be submitted soon, but I must be cautious about giving details.
Are you looking to commercialize the MegaBeacon work?
We were contemplating a patent this spring, but we could not do it because the doctoral student [working on it] had to finish a thesis. There was simply not enough time to go through the patenting process, so we had to leave that. So now it's out and public.
So basically anyone can use software to design their own MegaBeacon probes?
One can say that, yes. I actually have a special computer program [called Consort] that facilitates this, but that is not published yet, either. We work with that a lot and it helps us very much.
Is there anything else about this work that you think is worth noting?
People tend to accept target variability, for example in norovirus PCR, because they don't know there are other solutions, but now there is light at the end of the tunnel.
It could probably help a lot with norovirus PCR, because now people have to redesign each year their PCR for the current strain of norovirus, and with the MegaBeacon one could, I suppose, have much more lasting designs.