Comparison of low-density arrays, RT-PCR and real-time TaqMan® RT-PCR in detection of grapevine viruses

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Abstract

Low-density arrays (LDA) have been designed based on the real-time RT-PCR (TaqMan®) assays for the specific detection of 13 viruses that infect Grapevines in addition to the housekeeping gene 18S rRNA. The viruses included in the study are Grapevine leafroll associated viruses 1, 2, 3, 4, 5, and 9, Grapevine leafroll associated virus-2 Redglobe (GLRaV-2RG) strain, Ruspestris stem pitting associated virus, Grapevine vitivirus A, Grapevine vitivirus B, Grapevine fanleaf virus, Tomato ringspot virus (ToRSV), and Grapevine fleck virus (GFkV). This study includes three new TaqMan® RT-PCR assays that have been developed for GLRaV-2RG, GFkV and ToRSV and have been included in the TaqMan® RT-PCR and LDA detection. The LDAs were evaluated against a wide range of isolates distributed geographically. Geographical locations included Africa, Europe, Australia, Asia, Latin America and the United States. High-throughput detection of these viruses using LDAs was compared to RT-PCR and real-time TaqMan® RT-PCR. The efficiency of different RNA extraction methodologies and buffers were compared for use in low-density array detection. In addition improving the RNA extraction technique and testing the quality of the RNA using the 18S ribosomal RNA TaqMan® assay as an RNA specific internal control proved to generate better diagnostic assays. This is the first report on the use of LDA for the detection of plant viruses.

Introduction

Diagnostic tools for the detection of grapevine viruses have evolved through the years to include highly sophisticated and sensitive detection methodologies, starting from biological indexing using woody indicators and herbaceous hosts, enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR), and real-time TaqMan® RT-PCR. In biological indexing, the plant indicators for specific viruses are inoculated with samples taken from the candidate plants and observed for virus symptoms for a period of time. Biological indexing identifies the disease, but not the specific virus causing symptoms. For example, grapevine leafroll disease may be caused by one or multiple of eight viruses that have been reported to be associated with the disease (Martelli et al., 2002), and they show the same symptoms on the woody indicator host Vitis vinifera cv. Cabernet Franc. Other major limitations of the biological indexing are the time and the large greenhouse and field space requirements for its completion. Another widely used diagnostic method is ELISA which can be used to detect virus infection in grapevines (Rowhani, 1992, Frosline et al., 1996). Although this method is sensitive and reliable, it does have some disadvantages including its inability to detect grapevine viruses at low titer, absence of antibodies for some important viruses and difficulty in producing these reagents. PCR has been proven to be a more reliable method for the detection of grapevine viruses (Rowhani et al., 2000). The introduction of PCR has provided a new approach in grapevine virus detection because of its high sensitivity, specificity, and speed. The PCR is estimated to be 100–1000 times more sensitive than ELISA. However, PCR-based pathogen detection techniques suffer from inherent low throughput design. Moreover, mispriming and gel electrophoresis-based detection methods, respectively, decrease its specificity and efficiency. Real-time TaqMan® PCR detection is a very sensitive technique that can be used to overcome all the previous virus detection problems. Real-time PCR is increasingly being used for the detection and quantification of pathogens in plant tissue and soil (Agindotan et al., 2007, Lopez et al., 2006, Balme-Sinibaldi et al., 2006, Harju et al., 2005, Lunello et al., 2004, Mumford et al., 2000, Mumford et al., 2004, Osman and Rowhani, 2006, Osman et al., 2007, Schneider et al., 2004, Schaad and Frederick, 2002). TaqMan® or fluorogenic 5′-nuclease assays are based on the polymerase chain reaction, but also utilizes the novel fluorescent detection chemistry. During amplification the dual fluorescent labeled TaqMan® probe is cleaved by Taq DNA polymerase, separating the dyes and resulting in an increase in detectable reporter fluorescence in each cycle (Holland et al., 1991). This increase in reporter fluorescence corresponds to the amount of product amplified and is monitored in real-time during amplification. For some time, there were attempts to identify new technologies that can facilitate more qualitative and sensitive detection of multiple viruses in a single sample simultaneously. TaqMan® low-density arrays (LDA, Applied Biosystems, Foster City, CA, USA) have recently been introduced as a novel approach for pathogen detection. LDA is a modified method of real-time TaqMan® PCR that uses micro plates with 384 wells. Similar to real-time RT-PCR, these arrays enable a more focused and sensitive approach for the detection of plant pathogens while offering higher throughput compared to RT-PCR. In this study, the LDAs have been evaluated as a diagnostic tool for detecting grapevine viruses. Low-density PCR arrays using established protocols were developed by drying the real-time TaqMan® PCR primers/probes complexes into 384-well plates. Dried oligonucleotides of the TaqMan® primers and probes were reconstituted by adding cDNA mixed with PCR master mix and used as a custom low-density PCR array. LDAs while retaining the sensitivity of TaqMan® RT-PCR, allow the simultaneous quantification of large numbers of target genes (viral genomes) present in single samples. In the current study, the potential of LDAs for the detection of 13 different Grapevine viruses in infected tissues has been assessed. Key features of the LDA assessment included convenience, ease of use, rapidity, sensitivity and reproducibility.

Section snippets

Pathogens, plant material and reagents

Viruses used in this project included Grapevine leafroll associated viruses 1, 2, 3, 4, 5, and 9 (GLRaV-1, -2, -3, -4, -5, and -9), Grapevine leafroll associated virus-2 Redglobe (GLRaV-2RG) strain, Ruspestris stem pitting associated virus (RSPaV), Grapevine vitivirus A (GVA), Grapevine vitivirus B (GVB), Grapevine fanleaf virus (GFLV), Tomato ringspot virus (ToRSV), and Grapevine fleck virus (GFkV). All grapevine virus isolates were maintained in Vitis vinifera (grapevine) grown in the field.

Sample preparation

At the beginning of the experiments; leaf, petiole and cambial scraping samples were used in methods 1 and 2 and found that samples prepared from cambial scraping produced more consistent results, therefore, for further comparisons only samples from cambial scraping were used for total RNA preparation. In total RNA extraction methods comparison using LDA analysis, method 2 gave very poor RNA quality when evaluated by the 18S rRNA TaqMan® RT-PCR assay, therefore, this method was not included in

Discussion

Grapevines are perennial crops and vegetatively propagated either by grafting on different rootstocks or by self rooted cuttings. In grafted plants if either or both scion and rootstocks are infected with a virus or multiple viruses, the viruses transfer to the new plant. In addition, due to the relatively long life of the plants (several decades) they are exposed to different viruses transmitted by number of different natural vectors. As a result occasionally plants with multiple virus

Acknowledgements

This research was supported by the California Department of Food and Agriculture Fruit Tree, Nut Tree and Grapevine Improvement Advisory Board, American Vineyard Foundation, and California Competitive Grant Program for Research in Viticulture and Enology. We also thank the USDA National Clonal Germplasm Repository in Davis, CA for providing us with grapevine materials infected with different virus isolates.

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