Identification of Bacterial Plant Pathogens Using Multilocus Polymerase Chain Reaction/Electrospray Ionization-Mass Spectrometry

Abstract

Polymerase chain reaction/electrospray ionization-mass spectrometry (PCR/ESI-MS, previously known as “TIGER”) utilizes PCR with broad-range primers to amplify products from a 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 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 an 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 systems for agricultural diagnostic applications.

New Grower-Friendly Methods for Plant Pathogen Monitoring

“…Mass spectrometry can unambiguously measure the composition of nucleotides in PCR amplicons, providing sufficient information to identify microorganisms to the species level (29). Electrospray ionization-mass spectrometry has already been used to analyze the molecular mass of broad-range PCR products with a sufficiently high degree of precision to unambiguously derive base composition (99). Although the base order is not determined by this method, organisms can be accurately identified by matching base composition to an appropriate database.…”

Optical fiber-based synchronous fluorescence spectroscopy for bacterial discrimination directly from colonies on agar plates

“…36 For the same reason, spectroscopic methods have become a growing field of interest. Among these, mass spectroscopy 39 and infrared spectroscopy 8 have been well described. Fluorescence spectroscopy coupled with appropriate chemometric treatment of data has come to be considered as a powerful and fast tool of strain proximity for phylogenetic analysis as pointed out by Peix and colleagues in a recent review.…”

Outbreak of gastroenteritis caused byYersinia pestisin Afghanistan

“…All bacterial culture attempts from patients' blood and camel tissue were negative or only yielded contaminants, and few samples were available for testing ; the key to identifying the causative agent in this outbreak was the use of the Ibis T5000 PCR/ESI-MS assay. This diagnostic tool has previously been shown to detect a broad range of bacterial pathogens [16,17] and several viruses [18][19][20]. The inclusion of certain key gene targets in the Ibis T5000 biodefence assay allows it to detect and identify a wide range of pathogens in potentially complex clinical or environmental samples with little or no background effect.…”