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PNNLs Timothy Straub on Microarrays and Pathogen Detection

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AT A GLANCE
• Timothy Straub, Research scientist, analytical microbiology, Pacific Northwest National Laboratory.

Education
• Postdoc–Pacific Northwest National Laboratory
• PhD–Soil and Water Science, University of Arizona, 1993
• MS–Food Science, University of Arizona, 1991
• BS–Microbiology, University of Arizona, 1985

Timothy Straub has been involved in microarray research at Pacific Northwest National Laboratory since he started there as a postdoc in 2001. Now a research scientist, he has begun examining microarray applications in water-borne pathogen detection. He talked with BioArray News about his research and the hurdles his lab is looking to overcome to create a novel microarray-based detection device.

How are you using microarrays?

Traditional array experiments look at cells and their exposure to a particular chemical or stimulant, and at the up- and down-regulation of genes to see what changed, and what didn’t. What I try to do is look at microarrays in an entirely different way. This application of microarrays for detection of pathogens is very much in its infancy. There are a lot of publications that look at the detection of pathogen groups. What I’m waiting to see is more publications, and I want to write more about what we have seen, for true multi-agent pathogen detection in terms of nucleic acids. From the discussions I’ve had, there is an interest in multiplex nucleic acid detection because of the sensitivity and specificity of the methods.

I want to use the power of microarrays to look for pathogen threats in the environment and my particular interest is in pathogens in water, like the Norwalk-type viruses made famous on cruise ships, E. coli, and protozoans. While there is a whole genome array for E coli, that is not useful for me. I want to put the specific signatures of microorganisms like E. coli shiga toxin, [and] Norwalk viruses on a microarray. Wouldn’t it be useful to have all of those combined and have one assay that doesn’t depend on me knowing what I had already – one single assay that would allow me to take full advantage of one chip that could do it all?

Isn’t that a holy grail for microarrays?

Yes, it is, and honestly, I’m not there.

What do you lack?

To make an array that is useful, you would have to have a multiplex PCR system, where you could throw in all the different primers, amplify the environmental sample, and sort it out on the array. When you get up to six primer pairs, it gets difficult to start optimizing that reaction, and especially so when you go to 10-plex and up to 30-plex, incorporating fluorescent nucleotides becomes extremely difficult, due to the nature of the reaction.

You must have some solutions?

I do in my head. But, when you go to the bench, they kind of fizzle out. There are some solutions that are commercially available, that we [are] trying and evaluating. Qiagen has a PCR kit now that is optimized for a 16-plex PCR reaction and we will look at it this week. There is a group at Livermore National Lab who feels that it is the primer design that is impeding [the] multiplex PCR reaction and they are looking at bioinformatics software that will allow multiplex PCR.

We aren’t waiting for industry. We are trying to come up with our own solutions, some appear to have promise, and others appear to have promise on paper.

We are investigating different approaches to get away from PCR to solve multiplex problems. It’s very basic research that we haven’t validated. One method that we have tried [is] – we used a system put out by the Digene company where you detected RNA/DNA hybrids on the array. You take extracts from cells and hybridize total RNA to the array. Their system came in with an anitibody that specifically detected DNA/RNA hybridization. Then, you would come in with a labeled antibody. The idea was that at least for cells of interest, some genes are highly expressed, and if you can find those, you can avoid PCR altogether, and that would simplify the whole mess of multiplexing. We started working on it last year, and they pulled the product from the market, so we are stuck. That’s frustrating.

Oligos or cDNA?

I like oligos for the specificity that they provide. We use them at 25 to 40 nucleotides in length, and they provide a fair degree of specificity in the pathogens we’re looking for. I’m starting to experiment with more traditional cDNA products, trying to investigate RNA-based methods to detect RNA signatures from these pathogens. To do that, you need longer-length cDNA to be successful.

The RNA discovery process is particularly useful, and I could go with 70-mer or 130-mer lengths. But when you do that, we start running into the same issues of specificity that we are running into with the cDNAs, where we lose that specificity. For oligos, if you want exquisite specificity, I’ve been tending to use shorter, as opposed to longer. To pick up the mismatches, you need the shorter oligos – the longer ones tend to mask mismatches.

Do you already have libraries, and the preliminary work for cDNA available?

Our arrays have 50 signatures on them. That’s much easier to deal with than something that has 5,000 or 15,000 signatures on it. For me, cDNA vs. oligo costs are a wash. I know what specific genes I’m looking for and I can design the primers and do the PCR and I don’t have to have the high throughput that whole genome expression requires.

What do images from your arrays look like?

Processing is a lot more clear cut than typical expression data.

I’m looking for a specific gene, whether it is present or absent, on or off, and that’s easy to see. We have done a lot of work, and invested a lot of time in looking at specialized microarray software, and updating what we have, and writing our code. Where this really comes into play is in some of the work we do using microarrays to fingerprint organisms, moving from specific detection to taking isolates and saying can we tell the different isolates say of Xanthomonas. We are trying to get the database going from on vs. off to look at peak extraction protocols to say ‘Is that spot hybridized or not?’

Are there commercial products that would meet your needs?

There have been rumors of water-quality arrays in production: I’ve been hearing these about two or three years now. But, no vendor has come up to me and said ‘Would you be interested in beta testing some for us?’

There is a lot of interest in microarray-based detectors from a national security point of view. Certainly a system like the one you are researching might have such an application.

I believe that is why there is the amount of funding that is available for biodetection needs. To use the microarray as a smoke alarm-type of detector will take a lot of research. That’s a detect-to-warn system and for that, we need assays that can be done in minutes. Our problem right now is that we have to have a couple hours for PCR, and another couple of hours for hybridization.

What do you see as the end use of your microarray application: Will it sit in a pipeline somewhere?

The problem with an array itself is that it will not be useful for looking at water until you consider two other factors. One, you have to concentrate large volumes into tiny samples that can be assayed, and the second part is, if you concentrate a large water sample, then it is likely that there will be things that you don’t want to concentrate, like chemicals or other things that could perturb the hybridization assay. There is a need for a final product that will be hybridized to an array in a large enough sample that would be representative of what you would want to look at in water. We are starting to find better methods to concentrate a large enough volume of water that would be representative. I know other groups have looked at [sample collection and concentration] for air monitoring, but not that many people have looked at the water.

 

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