Poster Presentation Science Protecting Plant Health 2017

A novel isothermal DNA test for detection of Xylella fastidiosa (#230)

Rugang Li 1 , Keith Schuetz 1 , Bryant Davenport 1 , Paul Russell 1 , Alex Eads 1 , Shulu Zhang 1
  1. Agdia Inc, Elkhart, INDIANA, United States

Xylella fastidiosa is a gram-negative, xylem limited, bacterium that can be devastating to infected crops.  Although X. fastidiosa infects hundreds of plant species, it is commonly known as the causal agent of Pierce’s disease in grape, citrus variegated chlorosis (CVC), almond leaf scorch (ALS) disease, and olive quick decline syndrome (OQDS).   In Australia, even though the pathogen is not present, strict testing and quarantine requirements have been imposed on imported host plants to mitigate the risk of its introduction.  Testing for X. fastidiosa is commonly performed using ELISA or PCR methodologies.  PCR has historically offered the greatest level of sensitivity and specificity for X. fastidiosa but is laborious, taking hours to complete, and must be performed in a laboratory by skilled technicians.  Agdia, Inc has developed a rapid nucleic acid amplification assay, called AmplifyRP® XRT+, for detection of X. fastidiosa that is based on recombinase-polymerase amplification technology.  The test uses two target-specific primers and one internal probe that results in specific detection of X. fastidiosa. The test is performed in only 20 to 40 minutes at single operating temperature of 39°C using crude sample extracts.  Results can be visualized in real-time using a battery operated fluorometer or at end-point using a lateral flow strip housed inside a disposable amplicon detection chamber. 

The test was validated to detect multiple X. fastidiosa isolates collected from the U.S., Brazil, and Europe and demonstrated equivalent sensitivity when compared to published quantitative real-time PCR methods.  No false positives were observed when testing multiple host plants including grapevine, citrus, olive, blueberry and blackberry.  The validation data collected strongly supports this assay could be used as a reliable alternative to qPCR while offering the benefits of time savings, ease-of-use, and portability.