Meet Dr. Neil Boonham of the Food and Environment Research Agency (FERA)
Neil is a molecular virologist with over 16 year experience in the development and deployment of molecular diagnostics for a range of pathogens and pests, including the development of in-field diagnostics, next generation sequencing techniques and DNA barcoding methods.
“... I didn't really want to just go into a kind of blue-skies-academia. I was really interested in doing science that had a purpose in the short term.”
KLEMEN: Hello there! I am here with Dr. Neil Boonham. Neil, can you tell me a little bit why you are here and what are you doing in your life?
NEIL: So, I am here just giving a talk on diagnostic and detection methods at a qPCR (Real Time PCR) workshop. I'm a molecular biologist and plant pathologist from the Food and Environment Research Agency (FERA) in the UK and specializing in the development of diagnostics, both for use in the lab and also in the field, for a whole range of different target organisms: bacteria, insect, pest identification, viruses and fungi. The reason I do that is... well, the reason that it interests me is that, you know, when I left college, I didn't really want to just go into a kind of blue-skies-academia. I was really interested in doing science that had a purpose in the short term.
“If it doesn't work, they (the inspectors) are knocking on your door.”
I kind of like that kind of really quick feedback if you like and diagnostics development work has been really interesting to me, especially working somewhere like FERA where deployment of diagnostics is just as important as development. It's not just about doing the development right in a paper and having it sit on a shelf. The customers for your diagnostics are the inspectors and the diagnostic lab is, you know, in the same building. If it doesn't work, they're knocking on your door. So initially, that was what really interested me about that whole area, and that what’s kept me in it for so many years now.
“But the problem was always really the practicalities: keeping contamination under control, doing quantification effectively and routinely... qPCR on the other hand actually answered most of those questions... I mean it was real. I would say it was a real game changer for us in terms of a platform.”
KLEMEN: Alright, so how would you say qPCR is important for food industry? What kind of assays are you doing, what kind of value does it bring, the whole method, to the food industry?
NEIL: We started to work on qPCR, I suppose 15 to 20 years ago. Where previously we’ve been doing a lot of conventional PCR and you could see the potential in terms of sensitive and specific detection when compared with ELISA or other antibody based methods. But the problem was always really the practicalities, like keeping contamination under control, doing quantification effectively and routinely and also just the kind of work flow, you know, conventional PCR, running gels and that kind of interpretation of data of the gels just didn’t really seem suited, whilst you could use it in the research setting and it had potential, it didn’t really seem suited to a routine diagnostic lab.
qPCR on the other hand actually answered most of those questions. It enabled us to do quantification; it enabled us to do routine testing, including qualitative testing really in a straightforward fashion, which is very sensitive. You can tune the specificity to whatever you want. Yeah so, I mean it was real. I would say it was a real game changer for us in terms of a platform. It is also generic, so anything with the DNA on it is a target you can detect using that technique. And so for our lab, which works in lots of different areas across the agri-food chain, detection of pathogens for farmers right the way through to kind of food-safety-type work of detection of bacteria in food or GMOs in food. You know, you’ve got the same basic platform technology that you can use which again in terms of routine lab is fantastic. You don’t have to have a different method for each target or each application.You’ve got a platform technology you can use right the way across that is suited to high throughput testing too. So if you’re doing big surveys, than it’s also very suitable.
For most of what we’ve done over the last 10 to 15 years, whether it’s been in the routine lab, it’s gradually became a tool of the trade and it’s been the only technique really that’s pushing out some of the older techniques. So you can see now we tend to go straight to qPCR, as opposed to developing antibodies and developing new ELISA tests. This is the first technique that I’ve seen that’s really done that.
“I think one of the biggest problems that we have in our routine lab is, really, the use or the word “routine”. I think everybody then starts to just assume that nothing will ever go wrong.”
KLEMEN: We are here with Neil in Ljubljana, Slovenia, where Neil is giving a talk in the workshop (organized by BioSistemika) on plant pathology and qPCR in plant pathology, because Neil is an expert in troubleshooting qPCR. So, that being said, what kind of advice would you have for people out there that are using qPCR, maybe are starting to use qPCR and have troubles? No doubt everybody who starts using qPCR will eventually come to some sort of problems. Are there some general tips and advices you can give to people?
NEIL: Yes, I think... I mean we, like everybody, as you say, encounter problems. I think one of the biggest problems that we have in our routine lab is, really, the use or the word “routine”. I think everybody then starts to just assume that nothing will ever go wrong. “It’s routine, I don’t need to think about it too much”. And actually, you know, whilst these techniques are simple and routine to use you should always remember that any kind of test that you’re doing is effectively an experiment. You’re comparing your test sample against some controls using, in this case qPCR. In that sense it’s no different in terms of troubleshooting and problem solving than any other technique. You know, you’ve got to treat it like an experiment, use the right controls and interpret appropriately. I think... what we tend to find is, because we use the word “routine”, people tend to do a test: if it hasn’t worked just they repeat it without a second thought as to why it might have failed. So that’s quite common. And, you know, like anything else, just do good experiments and treat a test as an experiment.
“... people not willing to accept that they have contamination and trying to pin those events down to other artifacts...”
I think the other thing that’s really common was qPCR’s close tube system does control contamination pretty effectively. You will see contamination from time to time because it is incredibly sensitive and one of the things I’ve seen happen a lot in our lab is people not willing to accept that they have contamination and trying to pin those events down to other artifacts, say, of the process and invariably what you tend to find is (once you’ve gone through the troubleshooting) you’ve got contamination, just perhaps from an unexpected source. Perhaps just remember that contamination can happen all the way through from sampling through to DNA extraction, and critically during DNA extraction, sample setup, into running the instruments themselves so, again, include controls appropriately all the way through the process and not just assume that controls are something you’re going to do at the end and that you’re going to have a positive and negative control. It’s quite appropriate I think to run positive and negative controls all the way through those experiments. As I say, treat it as an experiment.
KLEMEN: qPCR has been now around for quite a few years, and there are new technologies coming up essentially every day: there were high hopes for microarray technology for example and it is nowadays scarcely used or is used for only specific applications. What do you see as the next big technology to replace qPCR? Is it NGS (Next Generation Sequencing), or perhaps a digital PCR, a special branch of NGS or something completely different? What is your opinion on that?
NEIL: Certainly from our experience there’s a range of different techniques around at the moment which I think will bring value into the lab in terms of Real-Time PCR, things like LAMP amplification (Loop Mediated Isothermal Amplification) seem to be pretty useful and you can run them on your Real-Time PCR instruments, so if you want to do really qualitative detection, presence/absence, but you want to do it much quicker than that chemistry seems to have a lot of legs on it and is quite useful. This isn’t at the moment particularly quantitative, although there is some work in the literature on people who are using it in a quantitative fashion.
“Clearly the other big one is NGS... you might be working in a lab that deals with the whole load of different problems and it’s a kind of platform technology that’s really suited to them.”
Clearly the other big one is NGS and its use in all kinds of different applications, and again, it’s this kind of generic platform technology, so you might be working in a lab that deals with the whole load of different problems and it’s a kind of platform technology that’s really suited to them. It could be looking at communities of pathogens or beneficial organisms, be they fungal, bacterial, mieofauna, that kind of thing, and that kind of metabolic coding approach. It could be quantitation and it could be transcriptomics and looking at gene expression or something like that. It could be a purely qualitative detection or characterization of new disease so you’ve got a whole range of applications that you can use a single platform technology for. So once you’re skilled up in doing the analysis and running the instruments you can use it for a whole range of different things. And that is not too dissimilar in terms of those gross characteristics to the way qPCR made a real impact in our lab too in the early days.
That kind of platform technology you could do lots of different things with. And then, I think also things like digital PCR. Relatively small modification of qPCR brings with it some of the benefits and advantages of qPCR but just changing the platform and the specific way it is working opens the door to more improved quantification, it’s easier to interpret quantification at the limit of detection which for a lot of applications is absolutely critical. So, again, another technique that I think... we are starting to see really good papers coming out in the literature in all sorts of different fields and another technique I think that in the next few years will really take off and become much more widely used and ultimately quite likely a tool of trade in routine diagnostic labs.
“... going from that early proof of concept of just being able to generate data that’s meaningful, all the way through to the point where you can do that in a reproducible fashion, takes a lot longer than you expect.”
KLEMEN: Coming back to the talk we had before about becoming the routine, how was this process for qPCR and do you see similar hurdles for these new technologies that are coming? I guess there is some lag between when the technology gets accepted as the gold standard before it is actually being routinely used on everyday basis without problems. What is your opinion on that?
NEIL: I think that’s an interesting point because you certainly see a new technique that takes off and what you see in the literature is a big explosion of papers coming out and you see almost every week a new paper and a new application of the technique. But then there seems to be this big lag phase between that happening and the method that’s being adopted in the routine lab. I think there are several different things going on there; I think one is the routine labs’ kind of conservative approach to taking on new technologies, which I don’t think is necessarily a bad thing. It ensures ultimately that they only take on techniques that are really robust enough to be in a routine lab. If they weren’t conservative and took on everything, they would get into a real, real mess really, so I think it is necessary. There’s that kind of aspect, on what’s happening.
“Well we’ve got this new method and I’ve done some validation, I’ve done some field validation, how much validation should I do? How much should it be compared with other methods?”
But then also, in our experience of developing techniques, going from that early proof of concept of just being able to generate data that’s meaningful all the way through to the point where you can do that in a reproducible fashion takes a lot longer than you expect. I think that also accounts for that kind of lag phase. And then I think in real routine diagnostics... I think availability of tests that are being validated to the appropriate standards, in our plant testing lab now we’re quite lucky that we’ve got things like an EPPO (European and Mediterranean Plant Protection Organization) validation standard for example, because before you kind of: ‘Well we’ve got this new method and I’ve done some validation, I’ve done some field validation, how much validation should I do? How much should it be compared with other methods?’ This kind of thing...
“It provides a framework that stops you from walking around in circles with a technique before it really gets adopted.”
So the more widespread use of things such as the EPPO validation standard and things like proficiency testing bring with them a framework of validation and an adoption of methods. I think that might help us to shorten that lag between that initial development and adoption. Because I don’t think there’s... it provides a framework that stops you from walking around in circles with a technique before it really gets adopted. I think there are a few different things going on and it’s not quite as straightforward to say it’s just about the science or the data, you know, there is a kind of social aspect of the labs as well as those kind of routine aspects that a lot of research labs don’t really worry about too much and then there’s another step there before adoption.
KLEMEN: That was Neil Boonham. We’re coming from Ljubljana, thank you Neil very much for your time and for sharing your thoughts.
NEIL: Thank you!
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