Development of Low Density PCR Array (LDA) for the Detection of Pathogens in Grapevines
The goal of this project was to develop sensitive and reliable molecular methods for the detection of viruses in grapevine. The method developed in this project is called “Low Density PCR Array” (LDA) and is a derivative of real time RT-PCR with 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 this project we also 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 (the buffer routinely used for the extraction of total RNA from different biological tissues by the automated 6700 Workstation) 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. Furthermore, the system capacity is approximately 400 samples per day and built into a BSL2 laminar flow hood to reduce cross contamination between samples. In an experiment, total RNA extraction methods were compared by using 29 vines infected with Grapevine leafroll associated virus (GLRaV) types 2, 3 or their combination. The RNA extraction methods used were Qiagene method and the 6700 automated workstation using 1X and 2X lysis extraction buffer. The TaqMan RT-PCR results showed 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.
Primers and TaqMan probes were also designed for 16 different viruses in grapevine and all were evaluated for their efficiency in detecting the viruses. These viruses included GLRaV-1 to -7, -9, Car; GLRaV-2-RG; Grapevine fanleaf virus (GFLV), Grapevine fleck virus (GFKV), Rupestris stem pitting associated virus (RSPaV); Grapevine virus A (GVA), B (GVB) and D (GVD). The LDA system developed for these 16 viruses was compared with TaqMan RT-PCR and the standard RT-PCR using 29 different grapevine cultivars and selections 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 could consistently detect the viruses in different growing season. The developed LDA method has been incorporated into testing scheme at Foundation Plant Services at UC Davis.