Potato (Solanum tuberosum) is the fourth most important global crop after wheat, rice and corn (FAO 2021). Diseases pose significant threat to the potato production such as yield reduction, quality decline, storage losses and increased costs. Therefore, a rapid and accurate diagnosis is essential for disease management. The quantitative real-time PCR (qPCR) technique is a highly effective molecular method that quantifies a specific target sequence of DNA or complementary DNA (cDNA) while providing real-time measurement of the fluorescence produced during each amplification cycle. Hence, qPCR is a widely used technique in plant disease diagnosis (Schaad and Frederick 2002; Miller et al. 2009). There are two types of qPCR techniques depending on if a fluorescent probe is involved: probe-based qPCR and SYBR-Green qPCR. In the probe-based qPCR diagnosis, primers are designed to be specific to the target pathogen’s genome, which ensures the qPCR reaction only amplifies the desired sequence but not the unrelated genetic material. In addition, the probe is also designed to be specific to the target pathogen’s genome and located between forward and reverse primers. The probe contains a fluorescent reporter and a quencher molecule. When the probe is intact, fluorescent signal is suppressed by the quencher; however, when the probe is cleaved by DNA polymerase during the extension of PCR, the quencher is separated with fluorescent reporter, leading to the increment of fluorescent signal that can be detected. In the SYBR-Green qPCR diagnosis, only primers are designed to be specific to the target pathogen’s genome and no probe is involved. The fluorescent signal is detected by the formation of double-stranded DNA, even there is no target amplicon. Therefore, probe-based qPCR has two advantages in plant disease diagnosis over SYBR-Green qPCR: i) higher specificity; ii) lower risk of false positive results.
In our routine diagnostic work, we found that some previous protocols could amplify products on non-target species (Feng, data not shown). In addition, previous studies indicated that PCR primers had tolerance to mismatches on templates even with four single nucleotide polymorphisms to the 3’ end of primers (Kwok et al. 1994; Christopherson et al. 1997; Klungthong et al. 2010; Green et al. 2015). Therefore, we believe it is necessary to test the specificity of previous protocols. In this study, specificity of 19 probe-based and four SYBR-Green based qPCR diagnostic protocols for 17 (six diseases included two qPCR diagnostic protocols) different potato diseases were tested by in silico analysis. The primers and probes (probe not applicable for SYBR-Green based qPCR) were subjected to BLASTn analysis against nucleotide collection (nr/nt) and whole-genome shotgun contigs (wgs) databases from NCBI to find the presence of the primer/probe sequences in non-target species. In the nr/nt BLASTn analysis, the sequence of the PCR product, i.e., from the 5’ end of the forward primer to the 3’ end of the reverse primer, was analyzed with default parameters. If sequence(s) identical or highly similar to the forward and reverse primers and probe sequence were found from any non-target hit, no more wgs analysis was conducted; Otherwise, wgs BLASTn analysis was conducted with “organism” limited on the genus level, e.g., Clavibacter michiganesis subsp. sepedonicus was analyzed on “Clavibacter (taxid:1573)” in which database the search was restricted to.
Double-stranded DNA fragments (gBlocks), primers and probes were synthesized by Integrated DNA Technologies (Coralville, IA). The gBlocks were dissolved and diluted according to instructions of manufacturer. The total volume of each qPCR reaction was 20 µL containing 1 µL DNA template regardless the DNA concentration, 0.2 µM of each primer, and 0.1 µM probe. The qPCR experiments were conducted in SsoAdvanced universal probe supermix (Bio-Rad Canada, Mississauga, ON) and consisted of an initial denaturation step at 95°C for 2 min, followed by 40 cycles of 5 s at 95°C and 1 min at 60°C. The qPCR experiments were repeated three times.
Probe-based qPCR diagnostic protocols
The specificity of 19 probe-based qPCR diagnostic protocols were tested, from which only 11 were highly specific to the target species including Clavibacter michiganesis subsp. sepedonicus (Schaad et al. 1999; Gudmestad et al. 2009), Synchytrium endobioticum (Van den Boogert et al. 2005), Ditylenchus dipsaci (Ponomareva et al. 2022, which developed two protocols), Globodera pallida (Gamel et al. 2017), Globodera rostochiensis (Gamel et al. 2017), Pratylenchus penetrans (Mokrini et al. 2013), Spongospora subterranea (Van de Graaf et al. 2003; Ward et al. 2004) and Potato mop-top virus (PMTV) (Pandey et al. 2020) (Table 1). However, eight showed high similarity to non-target sequences, which were Helminthosporium solani (Cullen et al. 2001), Candidatus Liberibacter solanacearum (Teresani et al. 2014), Streptomyces scabies and S. europaeiscabiei (Xu et al. 2016), Colletotricum coccodes (Cullen et al. 2002), Polyscytalum pustulans (Lees et al. 2009), Meloidogyne hapla (Sapkota et al. 2015), Phytophthora infestans (Lees et al. 2012), Potato spindle tuber viroid (PSTVd) (Boonham et al. 2004) (Table 1). Nucleotide polymorphisms of non-target sequence to primers and probes are listed in supplementary tables.
In vitro specificity test of H. solani qPCR protocol
In the probe-based qPCR diagnostic protocol for H. solani (Cullen et al. 2001), the primers/probe sequences had nine nt differences compared to their corresponding sequences in an alternative species H. velutinum (Genbank ID: MH151012.1) (Table 2). Two gBlocks of 180 bp (Fig. 1), containing the sequences the qPCR target in H. solani and H. velutinum, respectively, were synthesized to assess the protocol efficiency. The lowest number of gBlock molecules per reaction to generate a positive signal was 60 for H. solani and 600 for H. velutinum (Table 1b). From 600 to 600,000 molecules per reaction, the Cq values for H. solani were 3.46 lower than that of H. velutinum on average. Nevertheless, our data indicated that nine SNPs in the primers and probe cannot differentiate the two species by qPCR.
SYBR Green-based qPCR diagnostic protocols
The specificity of four probe-based qPCR diagnostic protocols were tested, from which none of them were specific to the target species including G. pallida (Madani et al. 2005), P. penetrans (Baidoo et al. 2017), P. infestans (Lees et al. 2012) and Ralstonia solanacearum (Chen et al. 2010) (Table 3). Nucleotide polymorphisms of non-target sequence to primers are listed in supplementary tables.