Issue link: http://read.dmtmag.com/i/81015
VINEYARD was caused by an artifact of the PCR process. TaqMan's other disadvantages compared to RT-PCR include the lim- itation in the availability of reliable probes for each virus and the signifi- cant expense to both develop and run. (The cost is even higher than RT-PCR and probes are not available to detect all known viruses.) Lastly, while both TaqMan and RT-PCR use two end primers, Taq- Man has an additional limitation because the probe must further match the virus template code in yet one more location. This increases the likelihood that TaqMan will miss virus detection in the event of virus mutation and yield a false negative result. This is a very practical con- cern since viruses in the vineyard have a high frequency of muta- tion. One can address the mutation issues by using multiple TaqMan probes (one for each mutation), but this fix only works for already char- acterized mutations. This under- scores that, regardless of RT-PCR or TaqMan, the issue of mutation is the critical reason why testing labs should include ELISA to comple- ment their grapevine virus testing program. While some researchers have stated that TaqMan is more sensi- tive than RT-PCR, differences in sensitivity are mostly a result of non-optimized RT-PCR procedures, especially in light of the fact that TaqMan and RT-PCR are variations of the same PCR technology. RESEARCH AND DISCOVERY METHODS Recently, grapevine viruses have been discovered and characterized using a technique called "deep" or "next generation" sequencing. A "genomic library" – a recombinant copy of all genetic material includ- ing grapevine nucleic acid – must be first produced from the individual grapevine (whether it is suspected to be infected or not) to be analyzed. The technique provides many genetic signatures (short sequenc- es) that are compared using com- puter algorithms. Because the grapevine genome is known, the host (grapevine) genes can be sub- tracted from the data so that the software only analyzes sequences that might represent viruses (or other pathogens). The researcher can focus the analysis on practi- cally any organism he or she wishes to study. The data output looks somewhat like a laundry list of known or related viruses and indicates the number of copies of each present. This method is very expensive (deep sequencing costs $8,000-$17,000 per run) and can only be applied to a few speci- mens at a time. Unexpected, yet interest - ing, results were published in the Liquid Nitrogen Dosing Systems: Unparalleled Precision Key Benefits: Flushes oxygen out of bottle head space Keeps the fruit in every pour Best "locks in" FSO2 levels www.chartdosers.com 800.371.3303 76 VINEYARD & WINERY MANAGEMENT SEPT - OCT 2012 ChartDosers HalfPg Wine_VWM.indd 1 WWW.VWM-ONLINE.COM 11/16/11 11:09 AM