Development of Low Density PCR Array (LDA) for the Detection of Pathogens in Grapevines.

The goal of this project is to develop a sensitive and reliable molecular method for the detection of viruses in grapevine. This method is called “Low Density PCR Array” (LDA) and is a derivative of real time RT-PCR using TaqMan probe (TaqMan RT-PCR). LDA system uses a fluorescently tagged TaqMan probe and it fluoresces in the presence of target viral RNA in an incremental level following each PCR cycle. The released fluorescent signal then is analyzed by a laser-based thermocycler and evaluated.

In the year 2006-2007 we have optimized the buffer for the 6700 automated Nucleic Acid Workstation for sample preparation and RNA extraction for use in LDA system. The results showed that the 2X ABI lysis buffer (from Applied Biosystems Inc.) worked better than 1X for grapevine tissue and it was comparable to the expensive and time consuming RNeasy column kit produced by Qiagen Inc. The 6700 automated system is quite fast and inexpensive and uses a 96 well plate format and can prepare RNA from 96 samples in approximately 2 hours. In an experiment we tested 29 vines by TaqMan RT-PCR for Grapevine leafroll associated virus (GLRaV) types 2 and 3 by comparing the 1X and 2X lysis buffer and the Qiagen system. In this experiment we found that 15, 12 and 19 plants were tested positive for GLRaV-2 by using Qiagen, 1X and 2X buffer, respectively. For GLRaV-3 the numbers tested positive were 9, 7 and 9 respectively. This comparison revealed that the 6700 automated system produces high quality RNA and was comparable to the RNA extracted by the Qiagen method.

We also designed primers and TaqMan probes for 14 different viruses in grapevine and all have been evaluated for their efficiency in detecting the viruses. These viruses included GLRaV-1 to -7 and -9; GLRaV-2-RG; Grapevine fanleaf virus (GFLV), Grapevine fleck virus (GFKV), Rupestris stem pitting associated virus (RSPaV); Grapevine virus A (GVA) and B (GVB). The LDA system developed for these 14 viruses was compared with TaqMan RT-PCR and standard RT-PCR using 29 different grapevine varieties with multiple virus infection from different grape growing regions in the world. The results showed that the LDA method was comparable to TaqMan RT-PCR in detecting viruses and both were superior to RT-PCR. The efficiency of RT-PCR, TaqMan RT-PCR and LDA in detecting viruses in different growing seasons were also compared using five vines each infected with GLRaV-1, -2, -3 and -5 (closteroviruses);GVA and RSPaV (rugose wood complex); GFLV (nepovirus); and GFkV (maculavirus). The results showed that TaqMan RT-PCR and LDA methods were more sensitive than RT-PCR and could consistently detect the viruses in different growing season.