TY - JOUR
T1 - Method
T2 - a single nucleotide polymorphism genotyping method for Wheat streak mosaic virus
AU - Rogers, Stephanie M.
AU - Payton, Mark
AU - Allen, Robert W.
AU - Melcher, Ulrich
AU - Carver, Jesse
AU - Fletcher, Jacqueline
N1 - Funding Information:
The authors acknowledge Jacob Price from Texas AgriLife Research Center, Mary Burrows from the Montana State University, Roy French from USDA-ARS, Geoffrey Dwyer from AGWEST plant laboratories in South Perth, Australia, TeeCie Brown from Oklahoma State University, and Trenna Bladgen from Oklahoma State University for supplying virus samples; Mike Palmer from Oklahoma State University and Andrew Doust from Oklahoma State University for their assistance with data analysis; and Mohammed Arif and Peter Hoyt from Oklahoma State University for proof reading the article. This work was approved for publication by the Director of the Oklahoma Agricultural Experiment Station and supported in part under project OKL 2052, and in part by USDA NIFA.
PY - 2012/5/17
Y1 - 2012/5/17
N2 - Background: The September 11, 2001 attacks on the World Trade Center and the Pentagon increased the concern about the potential for terrorist attacks on many vulnerable sectors of the US, including agriculture. The concentrated nature of crops, easily obtainable biological agents, and highly detrimental impacts make agroterrorism a potential threat. Although procedures for an effective criminal investigation and attribution following such an attack are available, important enhancements are still needed, one of which is the capability for fine discrimination among pathogen strains. The purpose of this study was to develop a molecular typing assay for use in a forensic investigation, using Wheat streak mosaic virus (WSMV) as a model plant virus.Method: This genotyping technique utilizes single base primer extension to generate a genetic fingerprint. Fifteen single nucleotide polymorphisms (SNPs) within the coat protein and helper component-protease genes were selected as the genetic markers for this assay. Assay optimization and sensitivity testing was conducted using synthetic targets. WSMV strains and field isolates were collected from regions around the world and used to evaluate the assay for discrimination. The assay specificity was tested against a panel of near-neighbors consisting of genetic and environmental near-neighbors.Result: Each WSMV strain or field isolate tested produced a unique SNP fingerprint, with the exception of three isolates collected within the same geographic location that produced indistinguishable fingerprints. The results were consistent among replicates, demonstrating the reproducibility of the assay. No SNP fingerprints were generated from organisms included in the near-neighbor panel, suggesting the assay is specific for WSMV. Using synthetic targets, a complete profile could be generated from as low as 7.15 fmoles of cDNA.Conclusion: The molecular typing method presented is one tool that could be incorporated into the forensic science toolbox after a thorough validation study. This method incorporates molecular biology techniques that are already well established in research and diagnostic laboratories, allowing for an easy introduction of this method into existing laboratories.
AB - Background: The September 11, 2001 attacks on the World Trade Center and the Pentagon increased the concern about the potential for terrorist attacks on many vulnerable sectors of the US, including agriculture. The concentrated nature of crops, easily obtainable biological agents, and highly detrimental impacts make agroterrorism a potential threat. Although procedures for an effective criminal investigation and attribution following such an attack are available, important enhancements are still needed, one of which is the capability for fine discrimination among pathogen strains. The purpose of this study was to develop a molecular typing assay for use in a forensic investigation, using Wheat streak mosaic virus (WSMV) as a model plant virus.Method: This genotyping technique utilizes single base primer extension to generate a genetic fingerprint. Fifteen single nucleotide polymorphisms (SNPs) within the coat protein and helper component-protease genes were selected as the genetic markers for this assay. Assay optimization and sensitivity testing was conducted using synthetic targets. WSMV strains and field isolates were collected from regions around the world and used to evaluate the assay for discrimination. The assay specificity was tested against a panel of near-neighbors consisting of genetic and environmental near-neighbors.Result: Each WSMV strain or field isolate tested produced a unique SNP fingerprint, with the exception of three isolates collected within the same geographic location that produced indistinguishable fingerprints. The results were consistent among replicates, demonstrating the reproducibility of the assay. No SNP fingerprints were generated from organisms included in the near-neighbor panel, suggesting the assay is specific for WSMV. Using synthetic targets, a complete profile could be generated from as low as 7.15 fmoles of cDNA.Conclusion: The molecular typing method presented is one tool that could be incorporated into the forensic science toolbox after a thorough validation study. This method incorporates molecular biology techniques that are already well established in research and diagnostic laboratories, allowing for an easy introduction of this method into existing laboratories.
KW - single nucleotide polymorphisms
KW - genotyping
KW - plant pathology
KW - viruses
KW - microbial forensics
KW - Single base primer extension
KW - SNaPshot Multiplex Kit
UR - http://www.scopus.com/inward/record.url?scp=84868572954&partnerID=8YFLogxK
U2 - 10.1186/2041-2223-3-10
DO - 10.1186/2041-2223-3-10
M3 - Article
C2 - 22594601
AN - SCOPUS:84868572954
SN - 2041-2223
VL - 3
JO - Investigative Genetics
JF - Investigative Genetics
IS - 1
M1 - 10
ER -