| Scope and method of study. The field of agricultural biosecurity combines traditional aspects of forensic science with plant pathology. With the threat of agroterrorism growing, steps must be taken in many areas to establish preparedness and to train enforcement personnel and extension agents in tracking and prosecuting the responsible individuals. The purpose of this study was to use Wheat Streak Mosaic Virus as a model system to develop a molecular assay using current, popular forensic laboratory equipment that can effectively compare samples collected from a suspected agroterrorism event against reference samples collected from a potentially responsible clandestine laboratory. Viral RNA extractions were performed with the MagMAX(TM) Kit (Ambion, Inc., Foster City, CA). cDNA synthesis and viral genome amplification were performed with the SuperScript(TM) One-Step RT-PCR Kit (Invitrogen, Inc., Carlsbad, CA). Single nucleotide polymorphisms were identified from sequencing data of three WSMV isolates. Three primers based upon these SNPs and a fourth primer included as an internal control and diagnostic marker were synthesized and the SNaPshot(TM) Kit (ABI, Inc., Foster City, CA) was used to discriminate the three WSMV isolates.; Findings and conclusions. The three SNP-specific primers used during SNaPshot(TM) analysis showed distinct qualitative differences between the three WSMV isolates tested. The fourth, internal control primer produced a positive result in every test, confirming the presence of WSMV cDNA within each sample. However, additional, unexpected peaks occurred at various sites in the electropherograms. After repeated SNaPshot(TM) reproducibility tests, blank assays, and negative controls, these additional peaks were determined to be evidence of mixed populations of WSMV within two of the three infections. The additional peaks therefore provided a second test for attribution that relied on the quantitative level of infection with genetically distinct isolates of WSMV instead of solely relying on the qualitative polymorphisms associated with an infection. This increased the potential discriminatory power of the assay exponentially. Future study with this assay would allow further testing of the reproducibility of the current isolates, subject previously uninvestigated natural isolates from around the world, and incorporate additional SNP-specific primers. |
