PCR Detects Plant Pathogens

Cepheid, Inc. (1190 Borregas Ave., Sunnyvale, CA 94089-1302; Tel: 408/541-4191, Fax: 408/541-4192; Website: www.cepheid.com) has entered into a Cooperative Research and Development Agreement (CRADA) with the U.S. Department of Agriculture (USDA) to develop DNA tests for rapid, field diagnosis of plant diseases threatening major cash crops. The tests will be developed and optimized for use on Cepheid's portable Smart Cycler TD and its GeneXpert systems.

Scientists with the USDA's Agricultural Research Service (ARS) will design DNA primers and probes to detect organisms responsible for several diseases posing serious threats, including citrus canker, Karnal bunt of wheat, Pierce's disease in grape vines, and plum pox in stone fruits. Cepheid retains first rights to license resulting diagnostic tests for commercial use on its systems. The company expects to supply the first samples of lyophilized, high- stability, high-performance testing kits for evaluation by the USDA during the first half of 2002.

Employing real-time polymerase chain reaction (PCR), Cepheid's portable systems are capable of detecting trace levels of DNA from disease-causing pathogens, including bacteria, viruses, and fungi, in 20 to 30 min. Until now, PCR tests have not been widely used for diagnosis of plant diseases, because they are typically performed on costly instruments available only in laboratories.

"Detection is our best line of defense against emerging plant pathogens," says Norm Schaad, a phytobacteriologist with the USDA's Foreign Disease-Weed Research Unit, who is heading up the project. "Making advanced testing methods like PCR cost effective and simple enough for widespread use could greatly improve our ability to quickly identify and respond to disease outbreaks."

Conventional detection methods, such as cell cultures and immunoassays, take between 2 and 12 days to provide results and are not able to detect most diseases in their early stages, before plants display symptoms.

The new DNA tests developed for use on the Smart Cycler TD will also operate on Cepheid's GeneXpert system, slated for availability in 2003. The GeneXpert system will automatically integrate all three steps required for PCR analysis-sample preparation, DNA amplification, and detection, enabling minimally trained operators to obtain a result from a raw sample in less than 30 min.

"Cepheid's systems are designed to take DNA testing out of the laboratory and put it to work in the real world, wherever it's needed," says Kurt Petersen, Cepheid's president and chief operating officer.

Citrus canker is responsible for the destruction of nearly 2 million trees in the state of Florida since the most recent infestation in 1995. Wheat fields in Texas, Arizona, and California are under USDA quarantine for Karnal bunt, a fungal disease that affects the quality of grain, limiting its uses and threatening crop exports. More than 100 million stone fruit trees in Europe are infected with plum pox, the most virulent strain of which was first discovered in the United States in 1999. With increasing numbers of insect carriers, Pierce's disease is a looming threat to California's $2.8 billion wine and grape industry. According to the Food and Agriculture Organization (FAO), annual U.S. crop losses due to all plant diseases are estimated at $9.1 billion.