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VINEYARD ficult to produce and good-quality reagents are not available for some of the important grapevine-infecting viruses such as Grapevine virus B (GVB). In recent years, the knowledge of grapevine virus genomic compo- sition has increased markedly, mak- ing RT-PCR a powerful technique for detecting grapevine viruses in vines with low-virus concentrations, which ELISA fails to do. The RT-PCR technique works by amplifying (i.e., making multiple copies of) the virus' genetic material extracted from the infected grapevines. The amplifi- cation process is repeated many times with each copy, making many more copies of the initial template. After the completion of the final cycle, over a billion copies of the virus are produced. Because most viruses infecting grapevines are composed of RNA, the viral RNA must first be con- verted into DNA prior to the ampli- fication step. A couple of primers, short pieces of DNA that specifi- cally bind to each side of the viral RNA, and an enzyme (Taq poly- merase) are required for the RT-PCR process. The virus-copied DNA is stained with a UV fluorescent dye and imaged using gel electrophore- ses, which separate the products by mass. If a fluorescent band of the correct size is detected, the pres- ence of the virus is confirmed. The RT-PCR method is laborious and expensive relative to ELISA – the cost of reagents for RT-PCR is roughly 10 times the cost for ELISA – and should be used for the detec- tion of viruses when concentration in the vine is uncertain. TaqMan is a specific type of PCR (also known as real-time or quanti- tative PCR). It is named after Ther- mus aquaticus, the bacterium, from which the Taq polymerase was isolated from, and its Pac Man-like chewing nature. This method is nearly identical to RT-PCR, except that a fluorescent- labeled probe (similar to the prim- ers described above) is included in the PCR reaction. During the ampli- fication process, each DNA copy releases a fluorescent signal from the probe. The total fluorescence is measured during each PCR amplifi- cation cycle in real time, hence its name "real-time RT-PCR" (TaqMan is the commercial name for a type of real-time PCR). Virus presence is concluded if a critical rise in fluorescence with each PCR cycle is detected. Taq- Man's primary advantage over RT-PCR is its ability to relatively quantify the concentration of virus rather than a yes or no of virus presence. However, since TaqMan cannot measure the amplified DNA fragment size, one can conclude a false POSITIVE virus presence when, by chance, the fluorescence WWW.VWM-ONLINE.COM SEPT - OCT 2012 VINEYARD & WINERY MANAGEMENT 75