Using in silico analysis to investigate the false positive potential of qPCR systems for potato disease diagnosis

Using in silico analysis to investigate the false positive potential of qPCR systems for potato disease diagnosis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Short Report Using in silico analysis to investigate the false positive potential of qPCR systems for potato disease diagnosis junye Jiang, Will Feindel, Michael Harding, David Feindel, Stacey Bajema, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3976832/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 02 Aug, 2024 Read the published version in European Journal of Plant Pathology → Version 1 posted 6 You are reading this latest preprint version Abstract Potato ( Solanum tuberosum ) is one of the most important global crops facing threats from different diseases. Rapid and accurate diagnosis is essential to control disease development and spread. Quantitative real-time PCR (qPCR) has been widely used in potato disease diagnosis. In this study, we evaluated the specificity of 19 probe-based and four SYBR Green-based qPCR protocols for 17 potato diseases using in silico analysis. Primers and probes of those protocols were subjected to BLASTn analysis against the nucleotide collection (nr/nt) database and the whole-genome shotgun contigs (wgs) database of NCBI for the presence of primer/probe sequences in non-target species. Results showed that 12 of 23 qPCR protocols were not specific to the target pathogens. A qPCR experiment indicated that even nine single nucleotide polymorphisms (SNPs) are present on the sequences of the primer/probe binding sites between the potato silver scurf pathogen Helminthosporium solani and its close-related species H. velutinum , the primers/probe specific to the former could amplify signals from the latter. These findings highlight the need for additional methods to enhance the diagnostic accuracy and new sequencing technologies such as next generation sequencing could provide useful information to develop specific diagnostic protocols for these pathogens. potato qPCR in silico SNPs Figures Figure 1 Introduction 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. Disscussion In conclusion, among the 19 probe-based qPCR diagnostic systems, 11 were specific to the target pathogens, but eight were not specific (Cullen et al. 2001 ; Teresani et al. 2014 ; Xu et al. 2016 ; Cullen et al. 2002 ; Lees et al. 2009 ; Sapkata et al. 2015; Lees et al. 2012 ; Boonham et al. 2004 ). Among the four SYBR-Green qPCR diagnostic systems, none was specific (Madani et al. 2005 ; Baidoo et al. 2017 ; Khan et al. 2017 ; Chen et al. 2010 ). In summary, 12 of 23 qPCR diagnostic systems were not specific to the target pathogens according to the in silico experiments. This is probably due to the limited genomic sequence information when these systems were developed. However, with rapid development of genomic sequencing technology such as next generation sequencing, the amount of genomic information of different species has been increased substantially. In all the systems not specific to the target pathogen, the system for H. solani detection (Cullen et al. 2001 ) was selected to test the amplification on H. velutinum sequence. The sequences of primers and probe had a total of nine SNPs to the corresponding sequences of H. velutinum , which is the biggest sequence variation between target pathogen and its close species among all the comparisons we conducted in this study. From 600 to 600,000 molecules, the Cq values of H. solani were 3.46 lower than that of H. velutinum on average, indicating not sufficient accuracy of specificity test even with 9 SNPs. For other non-specific primers, they have high potential to be false positive according to this study and previous work (Kwok et al. 1994 ; Christopherson et al. 1997 ; Klungthong et al. 2010 ; Green et al. 2015 ). Therefore, we recognize the need for additional methods to address the uncertainty and ensure a more reliable diagnostic approach, e.g., highly specific diagnostic system might need to be developed for identifying these 12 pathogens. In addition, the qPCR methods for disease diagnosis on other crops might need to be re-investigated based on updated genomic information. Declarations The authors declare no conflict of interest. Acknowledgements The authors thank Results Driven Agriculture Research (Grant number 2021G007R) to financially support this work. References Baidoo, R., Yan, G., Nagachandrabose, S., & Skantar, A. (2017). Developing a real-time PCR assay for direct identification and quantification of Pratylenchus penetransin soil. Plant Disease , 101 , 1432–1441. https://doi.org/10.1094/PDIS-01-17-0117-RE . Boonham, N., González Pérez, L., Mendez, M., Lilia Peralta, E., Blockley, A., Walsh, K., Barker, I., & Mumford, R. (2004). 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Detection and quantification of Spongospora subterranea in soil, water and plant tissue samples using real-time PCR. European Journal of Plant Pathology , 109 , 589–597. https://doi.org/10.1023/A:1024764432164 . Ward, L., Beales, P., Barnes, A., & Lane, C. (2004). A real-time PCR assay based method for routine diagnosis of Spongospora subterranea on potato tubers. Journal of Phytopathology , 152 , 633–638. https://doi.org/10.1111/j.1439-0434.2004.00908.x . Xu, R., Falardeau, J., Avis, T., & Tambong, J. (2016). HybProbes-based real‐time PCR assay for specific identification of Streptomyces scabies and Streptomyces europaeiscabiei , the potato common scab pathogens. Letters in Applied Microbiology , 62 , 153–159. https://doi.org/10.1111/lam.12522 . Tables Table 1 . In silico specificity test of probe-based qPCR diagnostic protocols Species Disease Protocol reference Risk of cross-reactivity Helminthosporium solani Silver scurf Cullen et al. (2001) H. velutinum Candidatus Liberibacter solanacearum Zebra chip Teresani et al. (2014) Ca . L. psyllaurous, Ca . L. africanus, Liberibacter crescens Clavibacter michiganesis subsp. Sepedonicus Ring rot Schaad et al. (1999); Gudmestad et al. (2009) NA Streptomyces scabies and S. europaeiscabiei Common scab Xu et al. (2016) S. griseiscabiei , S. caniscabiei , S. stelliscabiei , S. bottropensis , S. galbus Colletotricum coccodes Black dot Cullen et al. (2002) C. nigrum , C. incanum , C. dianense , C. gloeosporioides Polyscytalum pustulans Skin spot Lees et al. (2009) Helotiales sp., Tricladium sp., Cadophora sp., Mollisia sp. Synchytrium endobioticum Potato wart Van den Boogert et al. (2005) NA Ditylenchus dipsaci Stem nematode Ponomareva et al. (2022)* NA Globodera pallida Pale cyst nematode Gamel et al. (2017) NA Globodera rostochiensis Golden nematode Gamel et al. (2017) NA Meloidogyne hapla Northern root-knot nematode Sapkota et al. (2015) M. javanica Pratylenchus penetrans Root lesion nematode Mokrini et al. (2013) NA Phytophthora infestans Late blight Lees et al. (2012) P. andina , P. ipomoeae , P. urerae, P. citrophthora Spongospora subterranea Powdery scab Van de Graaf et al. (2003); Ward et al. (2004) NA Potato mop-top virus ( PMTV ) Pandey et al. (2020) NA Potato spindle tuber viroid (PSTVd) Boonham et al. (2004) Tomato chlorotic dwarf viroid , Rubber viroid India/2009 , Mexican papita viroid . *Two qPCR diagnostic protocols were developed in this reference. NA not applicable Table 2 . qPCR reactions on two gBlocks representing corresponding regions of H. solani and H. velutinum to the qPCR products OM967394.1 Molecule number 600,000 60,000 6,000 600 60 Cq value 22.63±0.05 26.37±0.02 29.89±0.02 34.23±0.21 37.41±0.51 MH151012.1 Molecule number 600,000 60,000 6,000 600 60 Cq value 26.24±0.05 29.66±0.11 33.27±0.07 37.77±0.43 NA Δ Cq 3.61 3.29 3.38 3.54 Table 3 . In silico specificity test of SYBR Green-based qPCR diagnostic protocols Species Disease Protocol reference Non-target amplification Globodera pallida Pale cyst nematode Madani et al. (2005) G. rostochiensis Pratylenchus penetrans Root lesion nematode Baidoo et al. (2017) P. fallax , P. convallariae Phytophthora infestans Late blight Lees et al. (2012) P. ipomoeae , P. phaseoli , P. andina , P. mirabilis Ralstonia solanacearum Bacterial wilt Chen et al. (2010) R. nicotianae , R. syzygii Supplementary Files Supplement.docx Cite Share Download PDF Status: Published Journal Publication published 02 Aug, 2024 Read the published version in European Journal of Plant Pathology → Version 1 posted Editorial decision: Major revisions 12 May, 2024 Reviewers agreed at journal 25 Feb, 2024 Reviewers invited by journal 25 Feb, 2024 Editor invited by journal 25 Feb, 2024 Editor assigned by journal 22 Feb, 2024 First submitted to journal 21 Feb, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3976832","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":274828632,"identity":"ce69f372-76ce-4549-b2ed-c84d121d581e","order_by":0,"name":"junye Jiang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA90lEQVRIiWNgGAWjYBAC9gYwJccDJBgfgNkSBLQwQrSwgbQwG5CkBUxIEKdlRu4xiQ8MbDL8s9uvVX6pOMzAP7uB8cMPvFry0iRnAB0mcedM2W2ZM4cZJO4cYJbswaslx0yaB+SXGzlptyXb0hgYbiSwMfAQ0vIHqEUeqKUYpEUeqIXxDx4tgiAtDAxuPAY30o8xfmyzYTAAamHGZ4s0zxtjyx6DHB7DGznM0gxnbHgM7xxslpbBo4WPPcfwxo8KC3u5G+kPP/6okJCTu9188OMbPFqAgEWCARyHPAYg9/DA4woPYP4AodkfMOKLjlEwCkbBKBi5AADnikTkzsqsvwAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0009-0002-8878-338X","institution":"Alberta Agriculture and Rural Development: Alberta Ministry of Agriculture Forestry and Rural Economic Development","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"junye","middleName":"","lastName":"Jiang","suffix":""},{"id":274828633,"identity":"456420d5-ce55-4dcf-bedc-22d81b63ee05","order_by":1,"name":"Will Feindel","email":"","orcid":"","institution":"Potato Growers of Alberta","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Will","middleName":"","lastName":"Feindel","suffix":""},{"id":274828634,"identity":"6a8472a8-7903-4aed-af3d-7eab35eeeda6","order_by":2,"name":"Michael Harding","email":"","orcid":"","institution":"Government of Alberta: Alberta Government","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Michael","middleName":"","lastName":"Harding","suffix":""},{"id":274828635,"identity":"ee22fb52-9e70-4915-8a96-6312cdfeb2f6","order_by":3,"name":"David Feindel","email":"","orcid":"","institution":"Government of Alberta: Alberta Government","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"David","middleName":"","lastName":"Feindel","suffix":""},{"id":274828636,"identity":"c1da321e-f9e9-4335-8197-61814ffdcf31","order_by":4,"name":"Stacey Bajema","email":"","orcid":"","institution":"Potato Growers of Alberta","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Stacey","middleName":"","lastName":"Bajema","suffix":""},{"id":274828637,"identity":"aae44802-71da-4a8b-9c46-db774e750372","order_by":5,"name":"Jie Feng","email":"","orcid":"","institution":"Government of Alberta: Alberta Government","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jie","middleName":"","lastName":"Feng","suffix":""}],"badges":[],"createdAt":"2024-02-21 21:09:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3976832/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3976832/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10658-024-02928-8","type":"published","date":"2024-08-02T15:56:55+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":51753117,"identity":"84cd74e1-9f8f-45f5-8d98-b98015d8cbe3","added_by":"auto","created_at":"2024-02-28 13:21:27","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":135515,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTwo gBlocks representing corresponding regions \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eH. solani \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eand \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eH. velutinum \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eto the qPCR products. Bold and italic letters, nucleotide polymorphisms in primers and probe; arrows indicate directions of primers and probe. *Identical nucleotide between \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eH. solani \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eand \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eH. velutinum\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3976832/v1/0e1953b58fb299da76304bfd.jpg"},{"id":61793278,"identity":"71ad497a-65eb-4332-b0d8-39f97923f680","added_by":"auto","created_at":"2024-08-05 16:09:01","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":636361,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3976832/v1/3f830295-537a-4300-865d-d023823ca052.pdf"},{"id":51753118,"identity":"6ee05a41-2f57-4227-ac50-f43b0f4a712d","added_by":"auto","created_at":"2024-02-28 13:21:27","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":51905,"visible":true,"origin":"","legend":"","description":"","filename":"Supplement.docx","url":"https://assets-eu.researchsquare.com/files/rs-3976832/v1/dc4282e9b9a74f81440c5e53.docx"}],"financialInterests":"","formattedTitle":"Using in silico analysis to investigate the false positive potential of qPCR systems for potato disease diagnosis","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePotato (\u003cem\u003eSolanum tuberosum\u003c/em\u003e) 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\u0026rsquo;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\u0026rsquo;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\u0026rsquo;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.\u003c/p\u003e\n\u003cp\u003eIn 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\u0026rsquo; 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 \u003cem\u003ein silico\u003c/em\u003e 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\u0026rsquo; end of the forward primer to the 3\u0026rsquo; 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 \u0026ldquo;organism\u0026rdquo; limited on the genus level, e.g., \u003cem\u003eClavibacter michiganesis\u003c/em\u003e subsp. \u003cem\u003esepedonicus\u0026nbsp;\u003c/em\u003ewas analyzed on \u0026ldquo;Clavibacter (taxid:1573)\u0026rdquo; in which database the search was restricted to.\u003c/p\u003e\n\u003cp\u003eDouble-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 \u0026micro;L containing 1 \u0026micro;L DNA template regardless the DNA concentration, 0.2 \u0026micro;M of each primer, and 0.1 \u0026micro;M 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\u0026deg;C for 2 min, followed by 40 cycles of 5 s at 95\u0026deg;C and 1 min at 60\u0026deg;C. The qPCR experiments were repeated three times.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProbe-based qPCR diagnostic protocols\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe specificity of 19 probe-based qPCR diagnostic protocols were tested, from which only 11 were highly specific to the target species including \u003cem\u003eClavibacter michiganesis\u003c/em\u003e subsp. \u003cem\u003esepedonicus\u0026nbsp;\u003c/em\u003e(Schaad et al. 1999; Gudmestad et al. 2009), \u003cem\u003eSynchytrium endobioticum\u0026nbsp;\u003c/em\u003e(Van den Boogert et al. 2005), \u003cem\u003eDitylenchus dipsaci\u0026nbsp;\u003c/em\u003e(Ponomareva et al. 2022, which developed two protocols), \u003cem\u003eGlobodera pallida\u0026nbsp;\u003c/em\u003e(Gamel et al. 2017), \u003cem\u003eGlobodera rostochiensis\u0026nbsp;\u003c/em\u003e(Gamel et al. 2017), \u003cem\u003ePratylenchus penetrans\u0026nbsp;\u003c/em\u003e(Mokrini et al. 2013), \u003cem\u003eSpongospora subterranea\u0026nbsp;\u003c/em\u003e(Van de Graaf et al. 2003; Ward et al. 2004) and \u003cem\u003ePotato mop-top virus (\u003c/em\u003ePMTV\u003cem\u003e)\u0026nbsp;\u003c/em\u003e(Pandey et al. 2020) (Table 1). However, eight showed high similarity to non-target sequences, which were \u003cem\u003eHelminthosporium solani\u0026nbsp;\u003c/em\u003e(Cullen et al. 2001), \u003cem\u003eCandidatus\u003c/em\u003e Liberibacter solanacearum (Teresani et al. 2014), \u003cem\u003eStreptomyces scabies\u003c/em\u003e and \u003cem\u003eS. europaeiscabiei\u0026nbsp;\u003c/em\u003e(Xu et al. 2016), \u003cem\u003eColletotricum coccodes\u0026nbsp;\u003c/em\u003e(Cullen et al. 2002), \u003cem\u003ePolyscytalum pustulans\u0026nbsp;\u003c/em\u003e(Lees et al. 2009), \u003cem\u003eMeloidogyne hapla\u0026nbsp;\u003c/em\u003e(Sapkota et al. 2015), \u003cem\u003ePhytophthora infestans\u0026nbsp;\u003c/em\u003e(Lees et al. 2012), \u003cem\u003ePotato spindle tuber viroid\u0026nbsp;\u003c/em\u003e(PSTVd) (Boonham et al. 2004) (Table 1). Nucleotide polymorphisms of non-target sequence to primers and probes are listed in supplementary tables.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIn vitro\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;specificity test of \u003cem\u003eH. solani\u003c/em\u003e\u003c/strong\u003e \u003cstrong\u003eqPCR protocol\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the probe-based qPCR diagnostic protocol for \u003cem\u003eH. solani\u003c/em\u003e (Cullen et al. 2001), the primers/probe sequences\u0026nbsp;had nine nt differences compared to their\u0026nbsp;corresponding sequences in an alternative species \u003cem\u003eH. velutinum\u0026nbsp;\u003c/em\u003e(Genbank ID: MH151012.1) (Table 2). Two gBlocks of 180 bp (Fig. 1), containing the sequences the qPCR target in\u0026nbsp;\u003cem\u003eH. solani\u003c/em\u003e and \u003cem\u003eH. velutinum\u003c/em\u003e, respectively, were synthesized to assess the protocol efficiency. The lowest number of gBlock molecules per reaction to generate a positive signal was 60 for \u003cem\u003eH. solani\u0026nbsp;\u003c/em\u003eand 600 for \u003cem\u003eH. velutinum\u003c/em\u003e (Table 1b). From 600 to 600,000 molecules per reaction, the Cq values for \u003cem\u003eH. solani\u003c/em\u003e were 3.46 lower than that of \u003cem\u003eH. velutinum\u003c/em\u003e on average. Nevertheless, our data indicated that nine SNPs in the primers and probe cannot differentiate the two species by qPCR.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSYBR Green-based qPCR diagnostic protocols\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe specificity of four probe-based qPCR diagnostic protocols were tested, from which none of them were specific to the target species including \u003cem\u003eG. pallida\u0026nbsp;\u003c/em\u003e(Madani et al. 2005), \u003cem\u003eP. penetrans\u0026nbsp;\u003c/em\u003e(Baidoo et al. 2017), \u003cem\u003eP. infestans\u0026nbsp;\u003c/em\u003e(Lees et al. 2012) and \u003cem\u003eRalstonia solanacearum\u0026nbsp;\u003c/em\u003e(Chen et al. 2010) (Table 3). Nucleotide polymorphisms of non-target sequence to primers are listed in supplementary tables.\u003c/p\u003e"},{"header":"Disscussion","content":"\u003cp\u003eIn conclusion, among the 19 probe-based qPCR diagnostic systems, 11 were specific to the target pathogens, but eight were not specific (Cullen et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Teresani et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Xu et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Cullen et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Lees et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Sapkata et al. 2015; Lees et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Boonham et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Among the four SYBR-Green qPCR diagnostic systems, none was specific (Madani et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Baidoo et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Khan et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Chen et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). In summary, 12 of 23 qPCR diagnostic systems were not specific to the target pathogens according to the \u003cem\u003ein silico\u003c/em\u003e experiments. This is probably due to the limited genomic sequence information when these systems were developed. However, with rapid development of genomic sequencing technology such as next generation sequencing, the amount of genomic information of different species has been increased substantially. In all the systems not specific to the target pathogen, the system for \u003cem\u003eH. solani\u003c/em\u003e detection (Cullen et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2001\u003c/span\u003e) was selected to test the amplification on \u003cem\u003eH. velutinum\u003c/em\u003e sequence. The sequences of primers and probe had a total of nine SNPs to the corresponding sequences of \u003cem\u003eH. velutinum\u003c/em\u003e, which is the biggest sequence variation between target pathogen and its close species among all the comparisons we conducted in this study. From 600 to 600,000 molecules, the Cq values of \u003cem\u003eH. solani\u003c/em\u003e were 3.46 lower than that of \u003cem\u003eH. velutinum\u003c/em\u003e on average, indicating not sufficient accuracy of specificity test even with 9 SNPs. For other non-specific primers, they have high potential to be false positive according to this study and previous work (Kwok et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; Christopherson et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Klungthong et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Green et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Therefore, we recognize the need for additional methods to address the uncertainty and ensure a more reliable diagnostic approach, e.g., highly specific diagnostic system might need to be developed for identifying these 12 pathogens. In addition, the qPCR methods for disease diagnosis on other crops might need to be re-investigated based on updated genomic information.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank Results Driven Agriculture Research (Grant number 2021G007R) to financially support this work.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBaidoo, R., Yan, G., Nagachandrabose, S., \u0026amp; Skantar, A. (2017). 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HybProbes-based real‐time PCR assay for specific identification of \u003cem\u003eStreptomyces scabies\u003c/em\u003e and \u003cem\u003eStreptomyces europaeiscabiei\u003c/em\u003e, the potato common scab pathogens. \u003cem\u003eLetters in Applied Microbiology\u003c/em\u003e, \u003cem\u003e62\u003c/em\u003e, 153\u0026ndash;159. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/lam.12522\u003c/span\u003e\u003cspan address=\"10.1111/lam.12522\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1\u003c/strong\u003e. \u003cem\u003eIn silico\u003c/em\u003e specificity test of probe-based qPCR diagnostic protocols\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eSpecies\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eDisease\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eProtocol reference\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eRisk of cross-reactivity\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eHelminthosporium solani\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eSilver scurf\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eCullen et al. (2001)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eH. velutinum\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eCandidatus\u003c/em\u003e Liberibacter solanacearum\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eZebra chip\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eTeresani et al. (2014)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eCa\u003c/em\u003e. L. psyllaurous,\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cem\u003eCa\u003c/em\u003e. L. africanus,\u003c/p\u003e\n \u003cp\u003e\u003cem\u003eLiberibacter\u0026nbsp;\u003c/em\u003ecrescens\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eClavibacter michiganesis\u003c/em\u003e subsp. \u003cem\u003eSepedonicus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eRing rot\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eSchaad et al. (1999); Gudmestad et al. (2009)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eStreptomyces scabies\u003c/em\u003e and \u003cem\u003eS. europaeiscabiei\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eCommon scab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eXu et al. (2016)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eS. griseiscabiei\u003c/em\u003e,\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cem\u003eS. caniscabiei\u003c/em\u003e,\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cem\u003eS. stelliscabiei\u003c/em\u003e,\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cem\u003eS. bottropensis\u003c/em\u003e, \u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cem\u003eS. galbus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eColletotricum coccodes\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eBlack dot\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eCullen et al. (2002)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eC. nigrum\u003c/em\u003e,\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cem\u003eC. incanum\u003c/em\u003e,\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cem\u003eC. dianense\u003c/em\u003e, \u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cem\u003eC. gloeosporioides\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003ePolyscytalum pustulans\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eSkin spot\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eLees et al. (2009)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eHelotiales\u0026nbsp;\u003c/em\u003esp., \u003cem\u003eTricladium\u0026nbsp;\u003c/em\u003esp., \u003cem\u003eCadophora\u003c/em\u003e sp., \u003cem\u003eMollisia\u003c/em\u003e sp.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eSynchytrium endobioticum\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003ePotato wart\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eVan den Boogert et al. (2005)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eDitylenchus dipsaci\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eStem nematode\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003ePonomareva et al. (2022)*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eGlobodera pallida\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003ePale cyst nematode\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eGamel et al. (2017)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eGlobodera rostochiensis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eGolden nematode\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eGamel et al. (2017)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eMeloidogyne hapla\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eNorthern root-knot nematode\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eSapkota et al. (2015)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eM. javanica\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003ePratylenchus penetrans\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eRoot lesion nematode\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eMokrini et al. (2013)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003ePhytophthora infestans\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eLate blight\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eLees et al. (2012)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eP. andina\u003c/em\u003e,\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cem\u003eP. ipomoeae\u003c/em\u003e,\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cem\u003eP. urerae,\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e\u003cem\u003eP. citrophthora\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eSpongospora subterranea\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003ePowdery scab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eVan de Graaf et al. (2003);\u003c/p\u003e\n \u003cp\u003eWard et al. (2004)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003ePotato mop-top virus (\u003c/em\u003ePMTV\u003cem\u003e)\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003ePandey et al. (2020)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003ePotato spindle tuber viroid\u0026nbsp;\u003c/em\u003e(PSTVd)\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eBoonham et al. (2004)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eTomato chlorotic dwarf viroid\u003c/em\u003e,\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cem\u003eRubber viroid India/2009\u003c/em\u003e,\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cem\u003eMexican papita viroid\u003c/em\u003e.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e*Two qPCR diagnostic protocols were developed in this reference. NA not applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2\u003c/strong\u003e. qPCR reactions on two gBlocks representing corresponding regions of \u003cem\u003eH. solani\u003c/em\u003e and \u003cem\u003eH. velutinum\u003c/em\u003e to the qPCR products\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.619967793880837%\" valign=\"top\"\u003e\n \u003cp\u003eOM967394.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.77294685990338%\" valign=\"top\"\u003e\n \u003cp\u003eMolecule number\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.721417069243156%\" valign=\"top\"\u003e\n \u003cp\u003e600,000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.721417069243156%\" valign=\"top\"\u003e\n \u003cp\u003e60,000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.721417069243156%\" valign=\"top\"\u003e\n \u003cp\u003e6,000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.721417069243156%\" valign=\"top\"\u003e\n \u003cp\u003e600\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.721417069243156%\" valign=\"top\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.619967793880837%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.77294685990338%\" valign=\"top\"\u003e\n \u003cp\u003eCq value\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.721417069243156%\" valign=\"top\"\u003e\n \u003cp\u003e22.63\u0026plusmn;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.721417069243156%\" valign=\"top\"\u003e\n \u003cp\u003e26.37\u0026plusmn;0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.721417069243156%\" valign=\"top\"\u003e\n \u003cp\u003e29.89\u0026plusmn;0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.721417069243156%\" valign=\"top\"\u003e\n \u003cp\u003e34.23\u0026plusmn;0.21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.721417069243156%\" valign=\"top\"\u003e\n \u003cp\u003e37.41\u0026plusmn;0.51\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.619967793880837%\" valign=\"top\"\u003e\n \u003cp\u003eMH151012.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.77294685990338%\" valign=\"top\"\u003e\n \u003cp\u003eMolecule number\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.721417069243156%\" valign=\"top\"\u003e\n \u003cp\u003e600,000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.721417069243156%\" valign=\"top\"\u003e\n \u003cp\u003e60,000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.721417069243156%\" valign=\"top\"\u003e\n \u003cp\u003e6,000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.721417069243156%\" valign=\"top\"\u003e\n \u003cp\u003e600\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.721417069243156%\" valign=\"top\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.619967793880837%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.77294685990338%\" valign=\"top\"\u003e\n \u003cp\u003eCq value\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.721417069243156%\" valign=\"top\"\u003e\n \u003cp\u003e26.24\u0026plusmn;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.721417069243156%\" valign=\"top\"\u003e\n \u003cp\u003e29.66\u0026plusmn;0.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.721417069243156%\" valign=\"top\"\u003e\n \u003cp\u003e33.27\u0026plusmn;0.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.721417069243156%\" valign=\"top\"\u003e\n \u003cp\u003e37.77\u0026plusmn;0.43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.721417069243156%\" valign=\"top\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.619967793880837%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.77294685990338%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026Delta; Cq\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.721417069243156%\" valign=\"top\"\u003e\n \u003cp\u003e3.61\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.721417069243156%\" valign=\"top\"\u003e\n \u003cp\u003e3.29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.721417069243156%\" valign=\"top\"\u003e\n \u003cp\u003e3.38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.721417069243156%\" valign=\"top\"\u003e\n \u003cp\u003e3.54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.721417069243156%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3\u003c/strong\u003e.\u0026nbsp;\u003cem\u003eIn silico\u003c/em\u003e specificity test of\u0026nbsp;SYBR Green-based qPCR diagnostic protocols\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eSpecies\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eDisease\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eProtocol reference\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eNon-target amplification\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eGlobodera pallida\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003ePale cyst nematode\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eMadani et al. (2005)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eG. rostochiensis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003ePratylenchus penetrans\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eRoot lesion nematode\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eBaidoo et al. (2017)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eP. fallax\u003c/em\u003e,\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003cem\u003eP. convallariae\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003ePhytophthora infestans\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eLate blight\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eLees et al. (2012)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eP. ipomoeae\u003c/em\u003e,\u003c/p\u003e\n \u003cp\u003e\u003cem\u003eP. phaseoli\u003c/em\u003e,\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cem\u003eP. andina\u003c/em\u003e,\u003c/p\u003e\n \u003cp\u003e\u003cem\u003eP. mirabilis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eRalstonia solanacearum\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eBacterial wilt\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eChen et al. (2010)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eR. nicotianae\u003c/em\u003e,\u003c/p\u003e\n \u003cp\u003e\u003cem\u003eR. syzygii\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"european-journal-of-plant-pathology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ejpp","sideBox":"Learn more about [European Journal of Plant Pathology](http://link.springer.com/journal/10658)","snPcode":"10658","submissionUrl":"https://www.editorialmanager.com/ejpp/default2.aspx","title":"European Journal of Plant Pathology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"potato, qPCR, in silico, SNPs","lastPublishedDoi":"10.21203/rs.3.rs-3976832/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3976832/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePotato (\u003cem\u003eSolanum tuberosum\u003c/em\u003e) is one of the most important global crops facing threats from different diseases. Rapid and accurate diagnosis is essential to control disease development and spread. Quantitative real-time PCR (qPCR) has been widely used in potato disease diagnosis. In this study, we evaluated the specificity of 19 probe-based and four SYBR Green-based qPCR protocols for 17 potato diseases using \u003cem\u003ein silico\u003c/em\u003e analysis. Primers and probes of those protocols were subjected to BLASTn analysis against the nucleotide collection (nr/nt) database and the whole-genome shotgun contigs (wgs) database of NCBI for the presence of primer/probe sequences in non-target species. Results showed that 12 of 23 qPCR protocols were not specific to the target pathogens. A qPCR experiment indicated that even nine single nucleotide polymorphisms (SNPs) are present on the sequences of the primer/probe binding sites between the potato silver scurf pathogen \u003cem\u003eHelminthosporium solani\u003c/em\u003e and its close-related species \u003cem\u003eH. velutinum\u003c/em\u003e, the primers/probe specific to the former could amplify signals from the latter. These findings highlight the need for additional methods to enhance the diagnostic accuracy and new sequencing technologies such as next generation sequencing could provide useful information to develop specific diagnostic protocols for these pathogens.\u003c/p\u003e","manuscriptTitle":"Using in silico analysis to investigate the false positive potential of qPCR systems for potato disease diagnosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-28 13:21:17","doi":"10.21203/rs.3.rs-3976832/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revisions","date":"2024-05-12T22:32:49+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2024-02-26T00:57:08+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-02-25T08:29:53+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"European Journal of Plant Pathology","date":"2024-02-25T06:24:09+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-02-22T10:25:07+00:00","index":"","fulltext":""},{"type":"submitted","content":"European Journal of Plant Pathology","date":"2024-02-21T11:27:01+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"european-journal-of-plant-pathology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ejpp","sideBox":"Learn more about [European Journal of Plant Pathology](http://link.springer.com/journal/10658)","snPcode":"10658","submissionUrl":"https://www.editorialmanager.com/ejpp/default2.aspx","title":"European Journal of Plant Pathology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"582e7a0c-603c-48ef-9bdb-fc8b52132678","owner":[],"postedDate":"February 28th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-08-05T15:59:08+00:00","versionOfRecord":{"articleIdentity":"rs-3976832","link":"https://doi.org/10.1007/s10658-024-02928-8","journal":{"identity":"european-journal-of-plant-pathology","isVorOnly":false,"title":"European Journal of Plant Pathology"},"publishedOn":"2024-08-02 15:56:55","publishedOnDateReadable":"August 2nd, 2024"},"versionCreatedAt":"2024-02-28 13:21:17","video":"","vorDoi":"10.1007/s10658-024-02928-8","vorDoiUrl":"https://doi.org/10.1007/s10658-024-02928-8","workflowStages":[]},"version":"v1","identity":"rs-3976832","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3976832","identity":"rs-3976832","version":["v1"]},"buildId":"ApUGefWb6u5IBVtyqm6d5","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}