A Novel and Highly Inclusive Quantitative Real-Time RT-PCR Method for the Broad and Efficient Detection of Grapevine Leafroll-Associated Virus 1

Abstract

Grapevine (Vitis vinifera L.) is one of the most important crops in the world due to its economic and social impact. Like many other crops, grapevine is susceptible to different types of diseases caused by pathogenic microorganisms. Grapevine leafroll-associated virus 1 (GLRaV-1) is a virus associated with grapevine leafroll disease and it is considered at the national and European level as a pathogen that must be absent in propagative plant material. For this reason, the availability of specific, sensitive and reliable detection techniques to ascertain the sanitary status of the plants is of great importance. The objective of this research was the development of a new GLRaV-1 detection method based on a TaqMan quantitative real-time RT-PCR targeted to the coat protein genomic region and including a host internal control in a duplex reaction. To this end, three new GLRaV-1 full genomes were recovered by HTS and aligned with all sequences available in the databases. The method has been validated following EPPO standards and applied for the diagnosis of field plant material and transmission vectors. The new protocol designed has turned out to be highly sensitive as well as much more specific than the current available methods for the detection and absolute quantitation of GLRaV-1 viral titer.

Keywords: HTS; diagnostics; grapevine; leafroll; virus.

Figure 1

(a) Maximum-likelihood phylogenetic tree constructed by MEGA X using the sustitution model GTR + G + I with ten GLRaV-1 complete genomic sequences. Accession numbers and/or isolate names are indicated. The scale bar shows the number of substitutions per site. Bootstrap percentages (1000 resamples) are indicated on the branches. Green dots indicate the new GLRaV-1 genomic sequences obtained in this study. (b) Sequence similarity matrix showing the percentages of nucleotide indentity between nearly complete genomes of GLRaV-1 isolates.

Figure 2

Frequency of primers/probes mismatches in GLRaV-1 detection methods [29,30,35,36]. A score-based color code was created to visualize the mismatches, showing the variant frequency in each position: nucleotides are nonmarked (variant frequency < 5%), yellow (5–20%), orange (20–30%) or red (>30%). Nucleotides located in the four first positions from the 3′ end of the sequence are also colored in red.

Figure 3

Validation of a grapevine internal control in a GLRaV-1 duplex real-time RT-PCR assay. Three independent replicates of a GLRaV-1 infected plant extract diluted with healthy plant extracts were assayed. GLRaV-1 amplification plots are shown for singleplex (blue) and duplex (red) reactions. A negative control (black) is included. PEP: phosphoenolpyruvate carboxylase; D: undiluted plant extract. −1:10⁻¹ dilution. −2:10⁻² dilution. −3:10⁻³ dilution. −4:10⁻⁴ dilution.

Figure 4

Absolute GLRaV-1 quantitation standard curve. Cycle threshold (Ct) values obtained for three replicates of ten-fold serial dilutions of GLRaV-1 control transcripts are plotted. The mathematical equation of the standard curve used for quantification and the coefficient of correlation (R²) are indicated.