Location: Floral and Nursery Plants ResearchTitle: Detection of plant quarantine pathogen Ralstonia solanacearum r3b2 with portable POCKIT™ and BLItz® systems Author
|Di, Rong - Rutgers University|
|Zhao, Liming - Rutgers University|
|Levy, Laurene - Animal And Plant Health Inspection Service (APHIS)|
Submitted to: Journal of Plant Health
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/31/2016
Publication Date: 9/15/2016
Citation: Di, R., Huang, Q., Stulberg, M.J., Zhao, L., Levy, L. 2016. Detection of plant quarantine pathogen Ralstonia solanacearum r3b2 with portable POCKIT™ and BLItz® systems. Journal of Plant Health. 1(1):103.
Interpretive Summary: Certain strains of the bacterial pathogen Ralstonia solanacearum cause brown rot disease in potato, and are therefore classified as select agents and are not allowed to enter the U. S., Canada and Europe. To make sure that plants imported from other countries do not carry this particular bacterium, a fast, accurate and reliable method to detect it is needed. We developed two portable systems for detection of this bacterium, one based on nucleic acid and the other on protein. These two independent, portable systems have the potential to facilitate detection of this important plant bacterial pathogen at the ports of entry and in field settings.
Technical Abstract: Ralstonia solanacearum (Rs) race 3 biovar 2 (r3b2) is designated as a quarantine pathogen in many countries and additionally as a Select Agent in the United States. Rapid, sensitive and accurate detection methods are urgently needed. We report here the development of two portable platforms for r3b2 detection, the TaqMan probe qPCR (quantitative, real-time polymerase chain reaction)-based POCKIT™ and the Rs monoclonal antibody (MAb)-coupled sensor BLI (bio-layer interferometry)-based BLItz®. Our data indicate that combining the palm sized POCKIT™ with the previously published TaqMan-based qPCR primers and probe set RsSA2, which targets a non-phage r3b2-unique sequence annotated as a probable n-6 adenine-specific DNA methylase, can detect as low as 10 CFU of the r3b2 strain UW551 in 32 minutes. The RsSA2/POCKIT™ system can specifically detect heat-inactivated Rs r3b2 strains, and such cells in spiked geranium stem sections, as well as in UW551-infected symptomatic and asymptomatic geranium plants. Additionally, we demonstrate that the BLI-based BLItz® instrument with the Forsite Rs MAb, which is more reactive to UW551strain than other Rs species complex members, can detect as low as 2.5×105 CFU/ml of UW551 in spiked geranium extract in a few minutes. The Rs MAb/BLItz® system has comparable rapidity and sensitivity to the commercial ImmunoStrip® with the advantage of higher r3b2 specificity. Moreover, the Rs MAb-coupled sensors are sensitive to Rs r3b2 after three months of room temperature storage, although the detection sensitivity was reduced by 28%. These two independent, portable systems have the potential to facilitate Rs r3b2 detection at the ports of entry and in field settings.