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DIAG CHIP
Plant health diagnostics on a microchip

The Diag Chip consortium.
The Diag Chip consortium.
The EU requires Member and Acceding States to be competent in diagnosing almost 300 plant pathogens listed under plant health legislation. Causal agents are diverse, from nematodes to viruses, so a range of expensive equipment and expertise is required. A group of European researchers are assessing the possibility of using gene chip technology to combine diagnosis of all these pathogens in a single test. As a case study, the project is producing a microarray that tests for notifiable pathogens of the potato. A full protocol for its use will be tested by diagnostic laboratories.

TA wide range of organisms can attack plants, from viruses to insects. Those that are serious pests and diseases are quarantined under the EU Plant Health Directive which lists almost 300 pathogens. This legislation controls the passage of plant material between countries, and is designed to prevent introduction of new pests or infectious diseases that threaten agriculture and the wider environment. To implement the legislation, Member States and Acceding countries must be competent in diagnosing all the pests and pathogens, some of which are obscure. Currently, diagnosis requires a wide range of expertise, from traditional taxonomy to classic microbiology and ultra-modern molecular biology. In some cases, expensive specialised equipment is required.

The Diag Chip project, with a consortium of plant disease scientists from Germany, France, Spain and the UK, recognised the potential of new genetic techniques to combine diagnoses in a single test. The partners are involved in a three-year project to study the feasibility of creating a ‘diagnostic chip’ that covers all types of notifiable plant pathogen. “DNA and RNA, the nucleic acids that hold genetic information, unify all life,” explains project coordinator Ian Barker of the UK’s Department for Environment, Food and Rural Affairs. “So they can form the basis of a method to deal with all the different pathogenic organisms at once.”

A new potato chip

The technology exploited by the project is the DNA microarray. This is a series of unique nucleic acid sequences, or probes, usually held as spots on a glass microscope slide or chip. Because strands of nucleic acid will hybridise into double strands with corresponding sequences, single-stranded pieces can be used to detect the presence of particular sequences from any sample of biological material. Label the nucleic acid in your sample with fluorescent tags and wash it over the chip. Any spot on the chip containing a genetic sequence that is present in the sample will fluoresce.

As a test case, the project is creating a microarray for potato pathogens. “We want to assess the applicability of highly parallel testing,” says Barker. That means using a common method to test for many different organisms. “We chose the potato, because it is an important European crop, with a manageable list of pests and diseases that includes most types of organism.” The chip will cover 24 potato pests and diseases, including 15 viruses and virus-like organisms, six nematode worms, one fungus and two bacterial diseases.

Potential plagues

Many dangerous potato pathogens still reside in South America, from where the potato itself came. Historically, there has been little or no trade in potatoes from the New World to the Old World, so we have escaped many of its diseases. But breeding material is often imported from South America, and has to be carefully monitored in potato quarantine stations.

To create a microarray for potato pathogens, a unique piece of genetic sequence must be selected for each organism. Some of the organisms, like Potato virus Y, are well studied, and their sequences are available on public access databases. “We are using the bioinformatics capability at the University of York to do the number-crunching involved in identifying a distinctive sequence for each species,” says Barker. “Other organisms, like the obscure South American viruses, have never been extensively characterised, so we are sequencing their genomes ourselves.” The partners in the project have different areas of expertise – the French scientists are working on nematodes, the Spanish group on bacteria and the Germans are focusing on viruses.

The tiniest trace

The main technical challenge is making the test sensitive enough. “The problem with highly parallel testing is that it is not as sensitive as ‘high-throughput’ tests, in which you test a lot of samples but only for one thing,” Barker explains. In high-throughput testing, known nucleic acid sequences are amplified many times, so the test can detect tiny quantities from the original sample. The conventional method of amplification, PCR, has to be done separately for each different sequence. “We need a generic way of amplifying all the nucleic acid in the sample in a single process. Otherwise, our test will only work if large amounts of pathogen are present, which will severely limit its application.” The team are currently testing a novel strategy to solve this problem.

Six routine testing laboratories around Europe have agreed to test the chip. “These are the people who hope to take up the technology,” says Barker. “To ensure that they feel comfortable with it, and that the methods we devise are practical, we have involved them right from the outset.”

“Microarrays have the potential to hold tens of thousands of sequences,” he continues. “If we can produce a chip for 23 potato pathogens, then it can certainly be done for the 300 pathogens in the EU Plant Health Directive.” The Diag Chip consortium includes a small Bavarian company, MWG, which brings a commercial perspective. “This may not be the final format,” Barker predicts. “Other microarray formats such as three-dimensional liquid arrays are emerging, and a huge investment is being made in miniaturisation and cost reduction. We are just proving the feasibility of highly parallel genomic testing for plant health.”

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