Being presented by Julie Pattemore
Molecular diagnostics utilising PCR techniques are routinely used for the detection and identification of pests in quarantine and biosecurity. When faced with making biosecurity decisions based on the finding of an unwanted organism by PCR, one of the major challenges is the inability to differentiate positive results originating from viable or dead cells. A positive PCR test is not sufficient to assess organism viability and traditional culturing techniques that could verify viability can be too time-consuming (1-30 days depending on pathogen and matrix). Viability PCR (vPCR) is a technique that selectively inhibits PCR amplification of DNA derived from dead cells, through the use of a nucleic acid intercalating dye. These dyes are unable to penetrate viable cells with intact membranes but will penetrate dead cells with compromised membranes. Exposure to blue light allows cross-linking of these dyes to DNA which inhibits PCR amplification.
The bacterial pathogen Xanthomonas fragariae can infect the calyx tissue associated with strawberry fruit. As fresh strawberries are a highly perishable commodity and X. fragariae is a slow-growing organism that can take over seven days to culture, a rapid method to detect viable cells is highly desirable. To enable rapid detection of viable X. fragariae, a vPCR protocol to discriminate viable and dead X. fragariae has been optimised. PEMAX™ (a double nucleic acid intercalating dye reagent) treatment resulted in complete inhibition of PCR amplification of 108-103 dead X. fragariae cells on strawberry host tissue. The most important parameters to optimise were amplicon length, turbidity of the sample matrix and choice of nucleic acid intercalating dye. This protocol will support timely decisions to be made if X. fragariae is detected on imported fresh strawberries.