New technique improves sensitivity of PCR pathogen detection

A new procedure devised by U.S. Department of Agriculture (USDA) scientists and colleagues can improve polymerase chain reaction (PCR)-based methods of detecting plant disease organisms.

PCR-based tests are prized tools for diagnosing plant diseases that can cause yield losses and diminished markets among other economic harm. But the test's ability to obtain a "genetic fingerprint" conclusively identifying a culprit pathogen hinges on there being a minimum number of its cells. Otherwise, the pathogen's genetic material can't be probed and multiplied in amounts necessary for detection, explains plant pathologist Norm Schaad, formerly with USDA's Agricultural Research Service (ARS). ARS is USDA's principal intramural scientific research agency.

Such diagnostic shortcomings can be especially costly when asymptomatic seed or plants intended for sale are certified as pathogen-free when, in fact, they are not, adds Schaad. He worked at the ARS Foreign Disease-Weed Science Research Unit in Frederick, Md., prior to retiring last year.

To tackle the problem, Schaad and colleagues Nikolas Panopoulos and Efstathios Hatziloukas devised a preliminary step called Bio-PCR. It uses growth-promoting agar or liquid media to increase the number of a target organism's cells in a sample prior to amplification of genetic material. In four to 72 hours, depending on the pathogen, the cells make thousands of new copies, enabling detection by direct PCR, according to Schaad.

Besides increasing sensitivity by 100- to 1,000-fold over conventional PCR methods, the enrichment technique stops substances called inhibitors from interfering with the action of a key enzyme, Taq polymerase.

Bio-PCR works best with fast-growing bacteria such as Ralstonia solanacearum, which causes bacterial wilt of potato and tomato, and Acidovorax avenae, which causes bacterial fruit blotch of watermelon. However, Bio-PCR also improves detection of slow-growing pathogens such as Xylella fastidiosa, responsible for Pierce's disease of grapes and leaf scorch of shade trees.

In studies with X. fastidiosa, Bio-PCR detected the bacterium in 90 percent of infected grape samples compared to 13 percent with conventional PCR methods.