Selection of an internal control (reference gene) for DiRT-qPCR in potato virus detection

To ensure correct potato virus detection, a constitutively expressed reference gene was needed for DiRT-qPCR assays. In quantitative detection, one or more reference genes are used for result normalization (Vandesompele et al. 2002; Bustin 2004; Huggett et al. 2005). Because DiRT-qPCR is mainly designated for qualitative potato virus detection, the internal control should work in the first-place as an amplification and extraction control for assurance of good assay performance and sample vitality. Therefore, the internal control needs to behave in crude potato sap as similar as possible to the target potato viruses. Besides spike-in controls (Huggett et al. 2005; Chen et al. 2016), plant innate housekeeping genes were often used as internal control genes (Nicot et al. 2005; González-Verdejo et al. 2008; Liu et al. 2018). Since the genome of all in this study investigated potato viruses (PVY, PVA, PVX, PLRV, PVS and PVM) are protein encapsulated, a protein encapsulated RNA spike-in control would full-fill all conceivable requirements.

However, spike-in controls are expensive and elaborative in production. Therefore, we decided to focus on comparison of two different TaqMan® _{primer sets detecting mRNA and/or DNA of}

Results and Discussion

oxidase and NADH dehydrogenase subunit 5, respectively. Since both housekeeping genes are coding for enzymes acting within the mitochondrion, all types of potato plant material are carrying genomic and transcriptomic nucleic acids of both housekeeping genes. For detection of cox DNA (plus mRNA), a TaqMan® _{primer set, published by Weller et al. 2000 was tested.}

For detection of mature nad5 mRNA, intron-spanning RT-PCR primers of Menzel et al. (2002) were exploited, which link exons a and b. Generally, immature nad5 mRNA shows an 848 bp intron (Kato et al. 1995) between exons a and b. Since the nad5 hydrolysis probe designed by Botermans et al. (2013) does not comply the specifications for DiRT-qPCR in terms of melting temperature, we tried to establish an appropriate hydrolysis probe for RT-qPCR detection for usage in combination with Menzel et al. (2002) intron-spanning nad5 primers (section 2.5.5, Table 7). Menzel et al. (2002) designed the nad5 detecting RT-PCR primers on the apple nad5 sequence (accession no. D37958), but the published intron-spanning primers were also effective for different potato-pathogen RT-PCR detection protocols (Seigner et al. 2008; Wei et al. 2009, Botermans et al. 2013). For hydrolysis probe design, unsuccessful search queries regarding potato derived nad5 sequences on NCBI (National Center for Biotechnology Information) and publications postulating a low sequence variability for plant nad5 (Souza et al. 1992) led us use the same nad5 apple sequence as Menzel et al. (2002) used for their primer design. According to Oligoanalyzer 3.1 of Integrated DNA Technologies (2018) the established probe nad5 _P_St14 (Table 7) has on default settings a Tm of 63,8 °C. Bustin

(2004) recommends for hydrolysis probes a 10 °C higher Tm than for forward and reverse

primers. Since the annealing and elongation temperature of the established potato virus DiRT- qPCR assays is 60 °C, we chose a Tm of 75 °C for the TaqMan® DiRT-qPCR probe detecting

nad5 mRNA. Therefore, we replaced four deoxycytidines at position 2, 9, 12 and 18 with C-5 propynyl-deoxycytidine modifications (Aschenbrenner et al. 2015; Figure 19 C), whereby every replacement increases the Tm of about 2.8 °C (Biomers.net 2018).

To compare the two TaqMan® _{primer sets with regard to their suitability as internal controls for}

DiRT-qPCR, we first tested the primer sets that amplify the expected nucleic acid types. Therefore, two different reaction compositions were tested for both internal control TaqMan®

primer sets. On the one hand, with addition of reverse transcriptase (RT) and on the other hand without reverse transcriptase (NRT). We homogenized a dormant potato tuber ('unk16') and used it as sample for DiRT-qPCR. Additionally, we used total RNA obtained from PLRV infected in vitro plants (1/045, Table 1). The extraction protocols is described in section 2.5.1.2. The reaction mix composition for the duplex DiRT-qPCR assay is listed in Table 12. The duplex reaction was conducted with either cox and PLRV or nad5 and PLRV detecting TaqMan®

primer sets (Table 7, Supplemental table 1). The thermal schedule for the reaction is listed in Table 13.

Figure 19 A presents the results for the internal controls in real-time and in end-point analysis. PLRV was detected in real-time and in end-point analysis as expected (data not shown). The nad5 fragment was strongest amplified in the RNA sample with RT addition. The RNA sample with the NRT condition, showed a slight fragment at nad5 size position in gel electrophoresis. The tuber sap sample did not show any amplification in real-time, nor in end-point analysis. All cox samples showed for both reaction compositions a respective sized fragment of about 69 bp in gel electrophoresis and an amplification curve in real-time.

Thereafter, we further compared in vitro plant sap and tuber sap samples in DiRT-qPCR assays regarding relative fluorescence unit (RFU) and Cq-values in real-time application and fragment intensity in end-point analysis to obtain a better insight to hydrolysis probe functionality and detection frequency of DNA and mRNA in case of the cox gene and nad5 transcripts. For nad5, the NTC did neither show any fragments in gel electrophoresis, nor a Cq-value less than the cut-off in real-time (Figure 19 B). However, we observed for nad5 gene strong fragment band severity in all tested potato samples, but we found very low RFU values and high Cq-values in real-time application. In contrast, end-point analysis of all samples showed for the detection of the cox gene weak fragment intensity at the expected position, but compared to nad5 similar or lower Cq-values in real-time application (Figure 19 B). The NTC used in the detection of the cox gene showed a slightly smaller sized fragment than expected and the real-time revealed a Cq-value that just exceeded the cut-off.

In addition, we investigated and compared the performance of the nad5 transcripts detected by nad5 forward and reverse primer (Menzel et al. 2002) and the protein coated PVY (Schubert et al. 2007 modified in this study, Supplemental table 1) in crude plant sap matrix. Therefore, we tested different homogenization liquids through comparison of the behavior of virus and mRNA transcripts in homogenization of further potato plant material ('unk17' – 'unk20') together with ultrapure H2O, formamide/H2O dilutions and 100% formamide. The

homogenization technique is described in section 2.5.1.1.1 and pictorial in Figure 18. The 1:10 dilution of the centrifuged mixtures were used in separate DiRT-PCR assays for detection of either nad5 or PVY. The reaction mix is described in Table 10 and the temperature schedule is described in Table 11.

The agarose gel electrophoresis of DiRT-PCR in Figure 18 shows the controls as expected. The detection of nad5 and PVY was compared regarding fragment intensity that differed for the nucleic acid types depending on the respective homogenization solutions. Sample processing, using formamide gave strongest results for mRNA (nad5) and using sterile H2O

both nucleic acid types were detected in a moderate manner as also described in section 3.3.2, Figure 17. Therefore, we were able to confirm the exclusive amplification of nad5 mRNA in potato material by the intron-spanning primers designed by Menzel et al. (2002) and the

Results and Discussion

principal amplification of mRNA and DNA by cox primers (Weller et al. 2000). The slight fragment band for nad5 in samples without SuperScript® _{III reverse transcriptase have been}

designated as laboratory contamination. This is because genomic DNA of nad5 contains introns and therefore would lead for the intron-spanning primer set designed by Menzel et al. (2002) to a larger fragment size of about 1029 bp instead of 181 bp.

Figure 18: Different amplification vitality of nad5 mRNA and PVY in different tuber homogenization solutions.

Comparison of homogenization solutions for DiRT-PCR with crude tuber sap to detect the internal control nad5 (plant innate mRNA) and the viral gRNA PVY, in combination. Five greenhouse produced tuber samples ('unk12'-'unk15') were homogenized in the presence of sterilized water and 100% v/v formamide and applied to DiRT-qPCR detection of nad5 (mRNA, above) and PVY (encapsulated potato virus, below). The sample preparation process is displayed on the right.

The investigation of DiRT-qPCR assays regarding hydrolysis probe functionality and detection frequency of mRNA (nad5) and DNA + mRNA (cox), showed strong fragment intensity for nad5, but very low RFU and high Cq-values in real-time application. In contrast, end-point analysis of the cox gene showed weak fragment intensity at the expected gel position, but similar or earlier Cq-values in real-time application (Figure 19 B). This displays a weak binding

and high Cq-values. Since the Tm of the nad5 hydrolysis probe was calculated at 75 °C, no

problems with probe annealing was expected at amplification temperature of 60 °C. In the end, after BLAST analysis of the apple nad5 sequence (accession no. D37958) to the potato genome, we found a putative potato nad5 gene in the second chromosome of Solanum tuberosum mitochondrion (accession no. MF989954, at position 94487-95578 bp). Alignment of the apple nad5 sequence and the putative potato nad5 discovered a single nucleotide polymorphism (SNP) at the first C-5 propynyl-deoxycytidine modification (Figure 19 C) that would explain the weak real-time detection of nad5 amplification. This leads to lowered Tm and

low 3 ′ -end binding affinity, which is important for initiation of probe fragmentation. However, the application of the nad5 hydrolysis probe designed by Botermans et al. (2013) in combination with C-5 propynyl-deoxycytidine modifications at five positions within the probe sequence, could lead to better results (nad5_P_Bo13_modified in Figure 19 C).

Additionally, for DiRT-qPCR in crude potato plant sap matrix, we found differences for both investigated RNA targets (mRNA: nad5 and viral gRNA: PVY). The nad5 transcript was stronger amplified in samples homogenized in 100% formamide (Figure 18) and H2O/formamide dilutions (Figure 17). In contrast, PVY was stronger amplified in samples

homogenized with only ultrapure H2O (Figure 17 and Figure 18).

We found that DNA-based internal controls better represent potato virus template vitality in DiRT-PCR and DiRT-qPCR than instable mRNA based internal controls do. For further experiments, we chose cox TaqMan® _{primer set (Weller et al. 2000) as internal control.}

Results and Discussion

Figure 19: Selection of an internal control (reference gene) in DiRT-qPCR for potato virus detection.

A: nad5 vs. cox with and without reverse transcriptase, amplification of mRNA and/or DNA and comparison of respective amplification in tuber sap in real-time and end-point analysis B: Comparison of Cq values based on probes from cox primer set (Weller et al. 2000) and nad5 primer set (established in this study, forward and reverse primer designed by Menzel et al. (2002) on RNA and tuber sap samples in real-time. End-point analysis based on forward and reverse primers of cox and nad5 primer set. C: The nad5 probe was designed based on nad5 sequence of Malus domestica (Accession no. D37958). An alignment of a putative nad5 nucleic acid sequence of Solanum tuberosum on chromosome 2 (Accession no. MF989954.1, starting at 94487 bases and ending at 95578 bases) with the apple sequence, revealed a SNP for the