|Baldwin, Carson - USAMRIID|
|Whitehouse, Chris - USAMRIID|
|Sampath, Ranga - IBIS, INC.|
|Harpin, Vanessa - IBIS, INC.|
|Li, Feng - IBIS, INC.|
|Melton, Rachael - IBIS, INC.|
|Blynn, Larry - IBIS, INC.|
|Drader, Jared - IBIS, INC.|
|Hofstadler, Steve - IBIS, INC.|
Submitted to: Phytopathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: May 1, 2008
Publication Date: November 1, 2008
Citation: Postnikova, E.N., Baldwin, C., Whitehouse, C., Sechler, A.J., Schaad, N.W., Sampath, R., Harpin, V., Li, F., Melton, R., Blynn, L., Drader, J., Hofstadler, S., Schneider, W.L. 2008. Identification of bacterial plant pathogens using multilocus PCR and electrospray ionization-mass spectrometry (PCR/ESI-MS). Phytopathology. 98-11-1156-1164 Interpretive Summary: Identification of bacteria from plant samples (phytobacteria) can be done by numerous methods, including PCR and antibody-based methods. However, most of the methods used for the detection of plant bacteria are only capable of detecting one species at a time. There are a few methods (called multiplex assays) that can detect up to 4 or 5 species of bacteria simultaneously. In addition, all of the techniques used to detect and identify plant bacteria require characterization and/or knowledge of the genetic sequence. PCR/electrospray ionization-mass spectrometry (PCR/ESI-MS, previously known as “TIGER”) is a newly developed assay with the capability to detect any and all bacteria in a given sample, including unknowns with no previous characterization. PCR/ESI-MS uses PCR reactions designed to generate amplified DNA from all bacteria followed by careful weighing of the PCR products using mass spectrometry. Computer analysis of the mass spectrometry is used to identify all of the bacteria in the sample by comparison of PCR product weights (and base contents) to a database. This technique is highly sensitive (similar to other PCR based assays), is high throughput and relatively cost effective. In initial tests using blinded panels PCR/ESI-MS successfully identified 93 percent of unknown bacterial DNAs to the genus level and 73 percent to the species level. The assay also successfully identified multiple bacteria in the same sample, and correctly identified bacteria from whole plant extracts.
Technical Abstract: PCR/electrospray ionization-mass spectrometry (PCR/ESI-MS, previously known as “TIGER”) utilizes PCR with broad range primers to amplify products from wide array of organisms within a taxonomic group, followed by analysis of PCR amplicons using mass spectrometry. Computer analysis of precise masses allows for calculations of base compositions for the broad range PCR products, which can then be compared to a database for identification. PCR/ESI-MS has the benefits of PCR in terms of sensitivity and high throughput capacity, but also has the distinct advantage of being able to detect and identify organisms with no prior characterization or sequence data. Existing broad range PCR primers, designed with the emphasis on human pathogens, were tested for their ability to amplify DNA of well characterized phytobacterial strains, as well as to populate the existing PCR/ESI-MS bacterial database with base counts. In a blinded panel study, PCR/ESI-MS successfully identified 93% of unknown bacterial DNAs to the genus level and 73% to the species/subspecies level. Additionally, PCR/ESI-MS was capable of detecting and identifying multiple bacteria within the same sample. The sensitivity of PCR/ESI-MS was consistent with other PCR based assays, and the specificity varied depending on the bacterial species. Preliminary tests with real life samples demonstrate a high potential for using PCR/ESI-MS system in the agricultural arena.