Grapevines can be infected with more than 60 viruses, of which many cause serious economic losses (Maliogka et al. 2015; Martelli 2014; Oliver and Fuchs 2011; Thompson et al. 2014). Most of the grapevine viruses contain positive sense single-stranded RNA genome (Maliogka et al. 2015; Martelli 2014) although recently some DNA viruses have also been described (Al Rwahnih et al. 2013; Basso et al. 2015; Zhang et al. 2011).

Since there is no environmentally safe and effective method to control virus diseases, the use of healthy planting stocks has a key importance to prevent their dissemination by infected propagating material (OEEP/EPPO 2008). Therefore selecting pathogen-free stocks and producing healthy plants are basic prerequisites in viticulture. Such procedures should be supported by rapid, reliable and economically feasible diagnostic protocols. Among these methodologies, PCR-based methods are the most popular and widely used (see e. g., Bertolini et al. 2010; Gambino and Gribaudo 2006; Jooste et al. 2015; Komorowska et al. 2014; Nakaune and Nakano 2006; Osman et al. 2008). The first step of the molecular methods for the detection of RNA viruses and gene expression studies is the isolation of high purity RNA suitable for cDNA synthesis followed by validation of cDNA using a housekeeping gene of the host plant. For such internal controls several specific primers have been published, e. g., for the constitutively expressing 18S rRNA (AF321266, Gambino and Gribaudo 2006), or actin (AF369525, Jelly et al. 2012) genes.

Although these primers efficiently amplify the target sequences from several grape genotypes, they produce products of the same size both from genomic DNA and cDNA. Thus complete removal of genomic DNA with DNAse treatment is required for the sake of reliable validation of cDNA synthesis, although the detection of RNA viruses is not affected by the presence of genomic DNA. In order to simplify the process and validate the efficiency of virus detection a new set of primers has been designed for internal controls. The primers encompass a region of two or three introns in the grapevine phosphoenolpyruvate carboxylase gene, which has been previously showed to be suitable as a reference gene in quantitative RT-PCR (Borges et al. 2014). Because of the difference in the amplicon sizes caused by the excised introns, DNA sequences amplified from genomic and cDNAs can be clearly distinguished by agarose gel electrophoresis.

Altogether 24 rootstocks, table and wine grape genotypes were used for the experiments. The seven rootstocks included are V. berlandieri x V. riparia ‘5C’, V. berlandieri x V. riparia ‘SO4’, V. berlandieri x V. riparia ‘5BB’, (V. berlandieri x V. riparia) x V. vinifera ‘Georgikon 28’, V. riparia x V. cinerea ‘Börner’, V. berlandieri x V. rupestris ‘Ruggieri 140’ and V. berlandieri x V. rupestris ‘110 Richter’. The 17 V. vinifera cultivars tested were ‘Kövidinka’, ‘Sárfehér’, ‘Kunleány’, ‘Miklóstelep 7’, ‘Kadarka’, ‘Kék bakator’, ‘Juhfark’, ‘Neoplanta’, ‘Pintes’, ‘Zefír’, ‘Furmint’, ‘Esther’ (or ‘Anna’ in PepS reactions), ‘Muscat Ottonel’, ‘Welschriesling’, ‘Vulcanus’, ‘Zervin’ and ‘Piros bakator’. Rootstocks (except ‘110 Richter’) were grown in a greenhouse, the other cultivars were propagated in vitro.

Total nucleic acids were isolated from young leaves using a CTAB-based protocol according to Xu et al. (2004) with a slight modification (the first washing step was omitted to retain cytoplasmic nucleic acids, like virus nucleic acids). Approximately 50 mg of plant material was lysed in 1.0 ml of lysis buffer and after the organic extractions nucleic acids precipitated with isopropanol were finally redissolved in 200 μl of sterile water. Samples were checked by agarose gel electrophoresis followed by ethidium bromide (EtBr) staining. Since most grapevine viruses are RNA viruses, LiCl or DNAse treatment was not applied.

For control experiments we used the 18S rRNA specific primers published previously (Gambino and Gribaudo 2006). Additionally, we designed two phosphoenolpyruvate carboxylase (PPC3, VIT_212s0028g02180, AF236126.1) gene-specific primer pairs using the Primer3 program (Untergasser et al. 2012). They were designated as PepLfw/PepLrev encompassing three introns, with the sizes of 770 nt, 113 nt and 1124 nt, and PepSfw/PepSrev encompassing two introns with the sizes of 113 and 1124 nt (Electronic Supplementary Material S1, for schematic presentation see Fig. 1). Primer sequences and their relevant data are shown in Table 1.

Fig. 1
figure 1

Schematic representation of the Vitis vinifera phosphoenolpyruvate carboxylase gene (PPC3, VIT_212s0028g02180, AF236126.1). Introns are indicated by full lanes. Exons are in black, 5′ and 3′ prime untranslated regions are in grey. The approximate positions of the PepL and PepS primers are indicated by arrows

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Table 1 Primers used for experiments
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Revert Aid First Strand cDNA kit (Thermo Scientific, #K1622) was used for cDNA synthesis with random hexamer primers according to the instructions of the supplier. The same cDNAs made in two independent replicates were used for subsequent PCR analysis. Polymerase chain reactions were carried out with KAPA Taq polymerase (KAPA Biosystems, KK1015) as proposed by the enclosed protocol. The following cycling parameters were used: initial denaturation at 94 °C for 3 min followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 50-58 °C (Table 1) for 30 s, extension at 72 °C for 1 min, and final elongation at 72 °C for 3 min. PCR products were analysed by electrophoresis in 1.5 (w/v) agarose gel stained with EtBr.

Detection of RNA viruses (most grapevine viruses have RNA as genetic material) by PCR starts with RNA isolation followed by first strand cDNA synthesis. At this point primers specific for a host housekeeping gene are commonly used as internal controls. In our initial experiments we used primers specific to the 18S rRNA gene (Gambino and Gribaudo 2006). This primer pair produced excellent results with all the 24 grapevine nucleic acid samples tested. The size of the PCR products however was the same when the crude plant nucleic acid preparations (Fig. 2a) or their first strand cDNA products (Fig. 2b) were used as templates. Therefore amplicons originating from genomic DNA or cDNA could not be distinguished.

Fig. 2
figure 2

Amplification products from total nucleic acids before cDNA synthesis (a) and from their first strand cDNAs (b) using the 18S rRNA-specific primers. Sample order: M: Gene ruler (Thermo Scientific #SM0321), Ø: negative control without DNA, lanes from 1 to 24: are V. berlandieri x V. riparia ‘5C’, V. berlandieri x V. riparia ‘SO4’, V. berlandieri x V. riparia ‘5BB’, (V. berlandieri x V. riparia) x V. vinifera ‘Georgikon 28’, V. riparia x V. cinerea ‘Börner’, V. berlandieri x V. rupestris ‘Ruggieri 140’ and V. berlandieri x V. rupestris ‘110 Richter’, followed by V. vinifera cultivars ‘Kövidinka’, ‘Sárfehér’, ‘Kunleány’, ‘Miklóstelep 7’, ‘Kadarka’, ‘Kék bakator’, ‘Juhfark’, ‘Neoplanta’, ‘Pintes’, ‘Zefír’, ‘Furmint’, ‘Esther’, ‘Muscat Ottonel’, ‘Welschriesling’, ‘Vulcanus’, ‘Zervin’ and ‘Piros bakator’. Both sets of nucleic acid samples yielded the same size (844 bp, Gambino and Gribaudo 2006) of amplification products

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Thus we designed two new PPC3 gene-specific primer pairs, PepLfw/PepLrev and PepSfw/PepSrev (Table 1), surrounding two and three introns, respectively, in the V. vinifera phosphoenolpyruvate carboxylase gene (Electronic Supplementary Material S1, Fig. 1). The PPC3 gene has been chosen for an internal reference control since it showed stable expression under different conditions (Borges et al. 2014). PCR reactions carried out with the same total nucleic acid preparations (containing DNA and RNA) as templates produced only the expected large (2724 and 1594 bp, respectively) fragments from the genomic DNA. The longer 2724 bp fragment was weakly amplified and only after longer extension time (3 mins, data not shown). The first strand cDNA samples generated by reverse transcription of the same total nucleic acid preparations produced an additional smaller fragments (717 bp with PepLfw/PepLrev primers, and 357 bp with PepSfw/PepSrev primers) due to the excision of the intron sequences (Fig. 3). Thus the use of primers surrounding intron(s) in the reference gene clearly discriminate between the amplifications from remnant genomic DNA and cDNA.

Fig. 3
figure 3

Amplification products using the PepLfw/PepLrev and PepSfw/PepSrev primers. (a): Amplification of the 717 bp sequence from cDNAs using the PepLfw/PepLrev primers. (b): PCRs with the PepSfw/PepSrev primers using total nucleic acids yielded only an 1594 bp product. (c): From cDNAs generated from the same nucleic acid preparations the PepSfw/PepSrev primers amplified both the genomic 1594 bp, and more abundantly the coding 357 bp sequences. Sample order: M: Gene ruler (Thermo Scientific #SM0321), Ø: negative control without DNA, lanes from 1 to 24: are V. berlandieri x V. riparia ‘5C’, V. berlandieri x V. riparia ‘SO4’, V. berlandieri x V. riparia ‘5BB’, (V. berlandieri x V. riparia) x V. vinifera ‘Georgikon 28’, V. riparia x V. cinerea ‘Börner’, V. berlandieri x V. rupestris ‘Ruggieri 140’ and V. berlandieri x V. rupestris ‘110 Richter’, followed by V. vinifera cultivars ‘Kövidinka’, ‘Sárfehér’, ‘Kunleány’, ‘Miklóstelep 7’, ‘Kadarka’, ‘Kék bakator’, ‘Juhfark’, ‘Neoplanta’, ‘Pintes’, ‘Zefír’, ‘Furmint’, ‘Esther’ (or ‘Anna’ in PepS reactions), ‘Muscat Ottonel’, ‘Welschriesling’, ‘Vulcanus’, ‘Zervin’ and ‘Piros bakator’

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To confirm these results PCRs were also carried out with PepLfw/PepSrev (encoding a 2800 bp amplicon from genomic DNA and a 793 bp amplicon from cDNA), as well as with PepSfw/PepLrev (encoding an 1518 bp amplicon from genomic DNA and a 281 bp amplicon from cDNA) primer combinations. These primer combinations also generated the amplification of the expected products (Electronic Supplementary Material S2). Taken together, we have shown that the two forward and the two reverse PPC3 gene-specific primers amplified the corresponding regions from the 24 cDNA samples in all of the four possible primer combinations, yielding the expected sizes (793, 717, 357 and 281 bp) of amplification products. These results indicate that the genomic regions used for primer design are highly conserved in the tested 24 grapevine genotypes.

Next first strand cDNA synthesis was carried out with oligo dT and PPC3 gene-specific primers instead of random hexamers followed by PCR analysis of cDNAs with PepSfw/PepSrev primers. The expected 357 bp region was amplified from all of the tested 24 cDNA samples along with the larger 1594 bp product derived from genomic DNA (Electronic Supplementary Material S3).

To experimentally confirm the potential use of these primers as internal controls in virus detection we also tested the same cDNA samples for the presence of Grapevine rupestris stem pitting-associated virus (GRSPaV) using the 48 V/49C primers (Lima et al. 2006). Interestingly, in the tested rootstock samples GRSPaV was not detectable, while nearly all V. vinifera cultivars proved to be infected (Fig. 4). The 48 V/49C primers tested in the presence of PepLfw/PepLrev primers also yielded the expected amplification products (330 and 717 bp, respectively, Electronic Supplementary Material S4). In our previous studies, approximately 85% of more than 120 grapevine samples were found infected with GRSPaV (data not shown). These results are consistent with other reports (Gambino et al. 2012; Komorowska et al. 2014; Osman et al. 2008).

Fig. 4
figure 4

Detection of GRSPaV in grapevine cDNA samples with 48 V/49C primers. Sample order: M: Gene ruler (Thermo Scientific #SM0321), Ø: negative control without DNA, lanes from 1 to 24: are V. berlandieri x V. riparia ‘5C’, V. berlandieri x V. riparia ‘SO4’, V. berlandieri x V. riparia ‘5BB’, (V. berlandieri x V. riparia) x V. vinifera ‘Georgikon 28’, V. riparia x V. cinerea ‘Börner’, V. berlandieri x V. rupestris ‘Ruggieri 140’, V. berlandieri x V. rupestris ‘110 Richter’, followed by V. vinifera cultivars ‘Kövidinka’, ‘Sárfehér’, ‘Kunleány’, ‘Miklóstelep 7’, ‘Kadarka’, ‘Kék bakator’, ‘Juhfark’, ‘Neoplanta’, ‘Pintes’, ‘Zefír’, ‘Furmint’, ‘Esther’, ‘Muscat Ottonel’, ‘Welschriesling’, ‘Vulcanus’, ‘Zervin’ and ‘Piros bakator’

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Although the elimination of DNA from plant nucleic acid extractions would not be necessary for PCR assays in detection of RNA viruses, quality control of cDNA synthesis can be compromised by the genomic DNA content of the samples. Even if a LiCl precipitation step has been included in the RNA purification protocol (for such methods see e. g., Gambino et al. 2008; White et al. 2008) some trace amounts of DNA sufficient to produce positive PCR results may still remain in RNA samples (Electronic Supplementary Material S5). Thus RNA samples should be treated with DNAse prior to cDNA synthesis, then DNAse should be removed by heat treatment or organic extraction followed by repeated precipitation and washing steps. These steps require additional time and costs for RNA preparation.

Using primers that surround an intron (introns) helps to avoid the DNA elimination step since the presence of an amplification product smaller by the size of the intron compared to the amplicons originating from genomic DNA clearly validate the successful cDNA synthesis. Such type of primers can be used as internal controls in the process of grapevine virus detection by RT-PCR to avoid false negative results, and are probably well suited also for other crop plants where RNA viruses cause serious infections.