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VINEYARD UC Davis was performed with nine positive and one negative control. Later we expanded the study in our laboratory by running dilution series of more than 30 positive and nega- tive controls to determine if one technique would be better than the other in detecting very low (dilute) concentrations of virus. This was done by running Taq- Man and RT-PCR side by side with serial dilutions of known infected grapevine samples from full viral strength (1:1) to one ten-thou- sandth viral strength (1:10,000), limited to only those viruses for which TaqMan is developed. We found no significant difference between the virus concentration detection sensitivity for TaqMan and RT-PCR (Figure 1). Having confirmed the detec- tion sensitivity of both methods as equivalent, we proceeded to the practical goal of this work: How do TaqMan and a full test panel com- pare in the detection of viruses with "real life" vineyard samples? The essential component to any diagnostic technique is that it must work in the field and not solely in the lab. Therefore, testing many vineyard and nursery samples rath- er than a limited number of posi- tive or negative controls, is a more effective way to validate a method. To determine this we used field samples randomly chosen from samples submitted to Eurofins STA Laboratories from clients' plant material collected in the 2011- 2012 fall/winter season. Within the group of 251 samples tested, TaqMan failed to detect virus infec- tion in many of the samples com- pared to the combined RT-PCR and ELISA panel. Remarkably, TaqMan missed the detection of samples infected with GLRaV-1, GLRaV-2, GLRaV-3, GLRaV-4, GLRaV -5, GFKV, and GVB (Table 2). No difference in detection between the methods was found when testing for GLRaV-2 Red Globe (RG strain, GLRaV-7, GLRaV- 9, Grapevine virus A (GVA), and Grapevine virus D (GVD). The lack of detection of the above viruses by TaqMan can be explained simply by the variation of mutant virus populations pres- ent in vineyards. When grapevine viruses multiply they are continu- ously mutating and are thus prone to minor genetic changes. As an example, several strains of Grapevine leafroll associated virus- 3 are known to infect vineyards in single or mixed populations. Because TaqMan uses probes that are very specific to genetic sequences and can only detect the exact match, its specificity causes the probe to miss slight genetic changes in the targeted viruses. The increased detection of Euro- fins STA HealthCheck Panel relative to TaqMan, in addition to not requir- ing extra TaqMan probes, is that the panel uses two complementary detection techniques – ELISA with broad-spectrum detection, and RT- PCR with specific and sensitive detection capabilities – instead of relying on one test method alone. As we increase our knowledge on the biology of grapevine viruses and continue to characterize known and new viruses, each of the test methodologies discussed in this article will play an important role in vine sanitation on foundation blocks and commercial vineyards. While This shows TaqMan and RT-PCR comparative results of the dilution series of a sample infected with GLRaV-3. The left panel shows a chart plotting the fluorescent light intensity versus the number of PCR thermal cycles (TaqMan amplification plot). Each of the curves represents the amplification of each of the serial dilutions. (Note that the 1:1 curve appears much earlier than the more diluted treatments.) The line in the plot (threshold line) is the level of detection or the point at which a reaction reaches a fluores- cent intensity above background. The curve observed at the very right of the chart (1:1000) appears to be at the limit of detection with a CT (the cycle at which the sample reaches the threshold line) value of 39 out of 40. The right panel shows the photograph of a gel where the same sample was analyzed with RT-PCR. Each band represents amplified DNA fragments for each dilution treat- ment. Note that both TaqMan and RT-PCR equally detected virus for dilutions of 1:1 down to 1:1,000. RT-PCR shows a faint band at the 1:10,000 dilution treatment; the same diluted treatment was not detected by TaqMan. 78 VINEYARD & WINERY MANAGEMENT SEPT - OCT 2012 WWW.VWM-ONLINE.COM