Development and validation of a real-time PCR assay for bioforensic detection of Pseudomonas syringae pv. tomato and evaluating the impacts of stressors on the effectiveness of multiple-locus variable number tandem repeat analysis (MLVA) and multilocus sequence typing (MLST) in microbial forensics investigations

Scope and Method of Study:An assessment of U.S. natural security conducted after the anthrax mail attacks of 2001 suggested that the agricultural sector is vulnerable to bioterrorist threats. To prepare for investigation of such events, a national capability in microbial forensics and forensic plant pathology is needed. As part of this program forensic methods must be developed and validated for use with plant pathogens and environmental samples associated with agricultural settings. This work describes: (1) the development and rigorous validation of a real-time PCR assay for bioforensic detection of the model plant pathogen Pseudomonas syringae pv. tomato, and (2) evaluation of the ability of two common microbial strain-typing methods, multiple-locus VNTR analysis (MLVA) and multilocus sequence typing (MLST), to reliably type P.s. tomato that may have recently undergone evolution during repeated sub-culturing for one year in planta and under a variety of laboratory conditions.Findings and Conclusions:The P.s. tomato detection assay is highly reproducible, displays linear amplification of the target DNA from 10 ng to 10 fg, consistently detects 100 fg of target nucleic acid, and is specific to P.s. tomato . Linear amplification of the positive control plasmid was also observed. Results obtained during validation of the P.s. tomato assay provide a template for the development and validation of similar assays for plant pathogens of high consequence.The MLVA fingerprints and MLST profiles from sub-cultured P.s. tomato DC3000 remained consistent throughout the experiment indicating that the selected genetic markers did not change over time. Thus, the specific MLVA and MLST typing systems used in this experiment should be reliable if used in a forensics investigation involving P.s. tomato.Further research should be conducted in a field setting to ensure that the assays can reliably type P.s. tomato infecting plants in their natural environment. Additionally, for use in typing other high consequence plant pathogens care should be taken to ensure that stable genetic loci are chosen as certain pathogens may display faster rates of mutation than P.s. tomato