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Q&A: Thai Researchers Develop Array-Based Food Pathogen Testing System


By Justin Petrone

nitsara_.jpgName: Nitsara Karoonuthaisiri

Title: Microarray Laboratory Director, the National Center for Genetic Engineering and Biotechnology, Thailand

Professional background: 2004 - present, head, microarray laboratory, National Center for Genetic Engineering and Biotechnology, Bangkok, Thailand

Education: 2004 — PhD, chemical engineering, Stanford University; 1999 — BS, chemical engineering, Columbia University

Seeking to help food producers identify pathogens in their products, researchers across the globe are using antibody arrays to provide higher-throughput, multiplex tools as replacements or supplements for traditional food-testing technologies like culture or enzyme-linked immunosorbent assays.

In Thailand, researchers at the National Center for Genetic Engineering and Biotechnology (BIOTEC), part of the country's National Science and Technology Development Agency, have developed an antibody array to detect food-borne pathogens, and hope to soon make the platform available to Thai industrial customers.

Led by Nitsara Karoonuthaisiri, the head of BIOTEC's microarray laboratory, this team last year published an article in the journal Biosensors & Bioelectronics demonstrating proof of concept for detecting bacteria in milk using an antibody array. Now, the team is working on moving its chemiluminescence-based detection system to a more automated technology platform to make it more accessible for its partners.

Karoonuthaisiri discussed BIOTEC's food-testing endeavors last week at Select Biosciences' Microarray World Congress in South San Francisco, Calif. BioArray News caught up with her during the conference and the following is an edited transcript of that interview.

What is your background? How did you get involved with microarray technology?

I did my undergrad at Columbia University. I did my masters and PhD at Stanford. My background was all in chemical engineering. At Stanford I did use microarray technology to study Streptomyces, which is an antibiotics-producing bacterium. That's how I got involved in using microarray technology. After that, I moved back to Thailand to establish a microarray lab at BIOTEC.

What do you do there?

BIOTEC is a national research center where we do a lot of programs. In my lab, we are using array technology as a platform to serve different kinds of needs. Most of our programs are in agricultural research because Thailand is big in agriculture. In my lab, we are using arrays to look at black tiger shrimp, for example. Thailand is the number one world exporter of shrimp, and this species of shrimp is considered a premium product with a higher market price than other shrimp. So, we are trying to revive the black tiger shrimp industry because they have a lot of problems with disease outbreaks and early maturation in captivity. We try to understand the fundamental biology of those processes by using DNA microarrays and other technologies at our lab. So that's where we use DNA microarrays. We are also making antibody arrays as a diagnostic kit to detect food-borne pathogens.

Why did you start the food-testing project?

As I mentioned, Thailand is a leading agricultural producer and food exporter, but we are competing with China, for example, and other countries with lower labor costs. So we need to set our status as a premium exporter, which means that we have to have strict food safety controls and good quality food. We believe we have the technology to satisfy the needs of industry.

What technology is currently being used in food testing and what are its shortcomings?

Right now, they use conventional methods like culture. You get a sample of the food and then you place it on a medium to grow bacteria for days or weeks, as some bacteria grow faster than others. So, if you wait for a couple of days, you can see bacteria colonies growing on the plate. But the thing is that each day you wait, industry loses money with the storage of potentially contaminated food. Culture is just an example of one method. There are also enzyme-linked immunosorbent assays or PCR. Currently, there is no perfect method for detecting food-borne pathogens.

Usually the amount of contamination in food is very little. If there were millions of bacteria in there, then you could even smell it. But since they are looking for such a little amount of bacteria, companies have to go through a process called enrichment, meaning that you pick a sample, put it in media, and let it grow. Typically, a company has to do this anyway. We plan to take the enriched sample and put it on our system. The faster you can do it, the cheaper you can do it, the better.

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When you started to design your assay what were you trying to accomplish?

What we have developed is still proof of concept, it's still a prototype, and it is not being used in the market right now. But we are trying to test many food-borne pathogens at the same time. For example, in an ELISA each well can detect one single pathogen, whereas in these arrays you can detect many pathogens at one time. Take your milk for example. You might want to know if it is contaminated or if it contains Salmonella. In an ELISA, you would have to take your milk sample and put it in one well to test for E. coli and another well for Salmonella, for example. But with antibody arrays, we can detect several pathogens in one sample. And we are trying to multiplex it, so that we could test not only your milk, but my milk as well on one single chip. That's being developed right now.

Can you describe your assay?

It's very similar to an ELISA. Basically, with an ELISA, you did the assay in a well, and you use a lot of antibody. Antibodies are very expensive. What we do is we have a slide. On the slide you have many spots. These spots correspond to different antibodies. One could be an antibody against E. coli, another spot could be an antibody against Listeria and another could be an antibody against Salmonella. You can add the sample to the slide, and if your sample is contaminated with any one of these pathogens then it would get captured onto that particular spot. You also use horseradish peroxidase antibodies as detecting molecules. That is the secondary antibody that links with enzymes. So if, for example, the E. coli spot has E. coli captured, the captured molecules will bind to E. coli and produce a chemiluminescent signal. Then you can see which spots have become positive and tell what pathogen it is according to where you spotted it in the first place and if your food is contaminated with a particular pathogen.

How did you test it to make sure it works?

This was developed using a buffer, but we have spiked pathogens into different kinds of milk to see if we can detect it, and right now we are in the process of testing it using different kinds of food.

What kinds of food?

I can't say. We are working with several food companies and that is confidential. They want to keep their competitive advantage over other food producers.

Let's just say that you demonstrate that it works in other foods. How will it be adopted beyond your lab?

Luckily, this work has been done in collaboration with NanoAsia and NanoDetection tecehnology, private companies that produce machines that detect chemiluminscent signals. So, at BIOTEC, we try to make sure that our research becomes a product. We have partnered with the companies to see it through to the end. Right now, food testing is being mostly done in the labs of the food companies. With the current technology we have, we could easily replace or supplement their current technologies. It doesn't require a technician or special skills, so it's not much of an adaptation for the user. It's not a diagnostic that a clinician would require special training to make it idiot proof. This is a normal method. They have in many cases already used ELISAs before, so it would be for them even easier than an ELISA.

You mentioned that you are doing more work on the assay?

Well, for the first paper we published on this, the results were detected using X-ray film, which is really not convenient, because if a lab does not have a dark room, they can't develop it. That's why we partnered with the companies. They are based in Thailand and Tennessee and produce a system called the NanoDetector, which is based on technology from Oak Ridge National Laboratory in Tennessee. This machine can read the signals of our spots. The idea is that it can give a number for the signal, whereas X-ray film can only give you the positive or negative result. The NanoDetector can give you quantitative numbers. It scores the intensity. In the case of these pathogens, it's not that important because you can't even have one of these pathogens in the food. But if you were using the same method for cancer samples from cancer patients, the number might be crucial in terms of treatment and telling you the stage of the patient. So, the capacity of the machine is beyond what we need right now.

But going forward, you'll be using the machine.

Yes, because with the X-rays you need a dark room. That is messy. We just started with X-rays because it was the fastest, most conventional way to get our proof of concept accepted to journals and the scientific community. Right now, we are optimizing our assay to enhance sensitivity and reduce assay time.