Premium
Validation of Real‐time PCR Assays for Bioforensic Detection of Model Plant Pathogens
Author(s) -
James Mindy,
Blagden Trenna,
Moncrief Ian,
Burans James P.,
Schneider Katherine,
Fletcher Jacqueline
Publication year - 2014
Publication title -
journal of forensic sciences
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.715
H-Index - 96
eISSN - 1556-4029
pISSN - 0022-1198
DOI - 10.1111/1556-4029.12321
Subject(s) - pathovar , pseudomonas syringae , biology , nucleic acid , pathogen , real time polymerase chain reaction , microbiology and biotechnology , computational biology , virology , bacteria , gene , biochemistry , genetics , pseudomonadaceae , pseudomonas aeruginosa
The U.S. agricultural sector is vulnerable to intentionally introduced microbial threats because of its wide and open distribution and economic importance. To investigate such events, forensically valid assays for plant pathogen detection are needed. In this work, real‐time PCR assays were developed for three model plant pathogens: P seudomonas syringae pathovar tomato , X ylella fastidiosa , and W heat streak mosaic virus . Validation included determination of the linearity and range, limit of detection, sensitivity, specificity, and exclusivity of each assay. Additionally, positive control plasmids, distinguishable from native signature by restriction enzyme digestion, were developed to support forensic application of the assays. Each assay displayed linear amplification of target nucleic acid, detected 100 fg or less of target nucleic acid, and was specific to its target pathogen. Results obtained with these model pathogens provide the framework for development and validation of similar assays for other plant pathogens of high consequence.