Development of a Low Density PCR Array (LDPA) for 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” (LDPA) and is a derivative of real time RT-PCR using TaqMan probe (TaqMan RT-PCR). LDPA 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 LDPA system. The results showed that the 2X ABI lysis buffer (from Applied Biosystem Inc.) worked better than 1X for grapevine tissue and it was comparable to the expensive and time consuming RNeasy column kit procedure 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 their evaluation and optimization for use in LDPA system is in progress. 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). LDPA system was evaluated for two viruses, GLRaV-2 and GLRaV-3 and the results were compared with RT-PCR, TaqMan RT-PCR, ELISA and biological index on Cabernet Franc indicator host. In this experiment 29 grapevines were tested. The results showed that 23 plants were tested positive either for GLRaV-2 or -3 or both by LDPA method (using 2X lysis buffere for RNA preparation) and by TaqMan RT-PCR. However, 22, 13 and 12 of these plants were positive by RT-PCR, ELISA and biological index, respectively