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Evaluating RNA extraction methods for the detection of citrus viruses and viroids with RT-qPCR and HTS
Thesis (MSc)--Stellenbosch University, 2025.
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Stellenbosch : Stellenbosch University
2025
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| _version_ | 1867613801575088128 |
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| access_status_str | Open Access |
| author | Dippenaar, Madelein |
| author2 | Bester, R. |
| author_browse | Bester, R. Dippenaar, Madelein |
| author_facet | Bester, R. Dippenaar, Madelein |
| author_sort | Dippenaar, Madelein |
| collection | Thesis |
| dc_rights_str_mv | Stellenbosch University |
| description | Thesis (MSc)--Stellenbosch University, 2025. |
| format | Thesis |
| id | oai:scholar.sun.ac.za:10019.1/134602 |
| institution | Stellenbosch University (South Africa) |
| language | English |
| last_indexed | 2026-06-10T12:41:54.752Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from SUNScholar — Stellenbosch University Repository |
| publishDate | 2025 |
| publishDateRange | 2025 |
| publishDateSort | 2025 |
| publisher | Stellenbosch : Stellenbosch University |
| publisherStr | Stellenbosch : Stellenbosch University |
| record_format | dspace |
| source_str | SUNScholar — Stellenbosch University Repository |
| spelling | oai:scholar.sun.ac.za:10019.1/134602 Evaluating RNA extraction methods for the detection of citrus viruses and viroids with RT-qPCR and HTS Dippenaar, Madelein Bester, R. Maree, H. J. Stellenbosch University. Faculty of AgriScience. Dept. of Genetics. Citrus -- Diseases and pests Plant viruses -- Detection Viroids RNA -- Detection Polymerase chain reaction UCTD Thesis (MSc)--Stellenbosch University, 2025. Dippenaar, M. 2025. Evaluating RNA extraction methods for the detection of citrus viruses and viroids with RT-qPCR and HTS. Unpublished masters thesis. Stellenbosch: Stellenbosch University [online]. Available: https://scholar.sun.ac.za/items/2e22d0ea-fba3-494f-a055-dc494803ae26 ENGLISH ABSTRACT: The South African citrus industry is a vital economic role-player and certification, quarantine and disease management practices are reliant on accurate detection assays. Effective nucleic acid extraction is the cornerstone of various molecular detection techniques including traditional polymerase chain reaction (PCR) methods and high-throughput sequencing (HTS). The extraction method considerably influences the type, quantity and quality of RNA extracted and, therefore, the overall detection capability of the assay. As the popularity of HTS for application in diagnostics increases, it is imperative to evaluate nucleic acid extraction methods and to explore various sequencing methodologies to ensure that the entire pathogen detection pipeline is optimised. Illumina platforms have been extensively validated for this purpose but recently, Oxford Nanopore Technologies (ONT) has become a contending platform for integration. The continuous development of HTS necessitate the renewed comparison of sequencing platforms. This study aimed to identify an RNA extraction protocol that will enable the consistent, effective, and reliable detection of citrus virus and viroid RNA with reverse transcription quantitative PCR (RT-qPCR) and HTS. Overall five extraction protocols, cetyltrimethylammonium bromide (CTAB)/lithium chloride (CL), CTAB/ethanol (CE), acidic phenol buffer/lithium chloride/isopropanol (TL), sodium dodecyl sulphate (SDS)/isopropanol (SI) and CTAB/isopropanol (CP), were selected and pathogen quantities in citrus infected with citrus tristeza virus (CTV), citrus virus A (CiVA), citrus tatter leaf virus (CTLV), citrus dwarfing viroid (CDVd), citrus exocortis viroid (CEVd) and hop stunt viroid (HSVd), were evaluated with RT-qPCR and validated with Illumina HTS. Extracts from one protocol were then selected and used to compare Illumina and ONT virus and viroid sequencing capability. The extraction protocol had a significant influence on differential pathogen detection ability for both RT-qPCR and HTS. The CL method performed best for CTV while the SI method proved more sensitive for the other viruses. Viroid detection had increased variability with CE extracts containing more viroid RNA although CL extracts showed comparable amounts of CDVd. The CE protocol indicated potential debilitating effects on virus detection although a protocol modification showed increased virus and reduced viroid amount. Despite the differential pathogen extraction amongst protocols, all target pathogens could be detected with appropriate RT-qPCR replicates and combined bioinformatic approaches. This study emphasises that although multiple protocols can be effective, further selection and optimisation could enhance the performance. The SI extracts were selected to compare sequencing platforms and both technologies were able to identify all pathogens using both reference-dependent and independent methods. Illumina re-established its reputable high sensitivity, coverage and accuracy. ONT compensated for fewer pathogens reads and lower depth with longer reads that enabled reasonable genome coverage and sequencing identities comparable to that of Illumina when appropriate flow cell capacity (data amount) was used. This comparison between Illumina and ONT platforms highlights the strengths associated with each approach and offers new insights into the possible application of HTS within plant health surveillance and biosecurity programs. Present research serves as a foundation for the refinement of diagnostic pipelines in citrus pathology. AFRIKAANSE OPSOMMING: Die Suid-Afrikaanse sitrusbedryf is van groot ekonomiese belang, en sertifisering-, kwarantyn- en siektebestuurspraktyke is afhanklik van akkurate opsporingstoetse. Effektiewe nukleı̈ensuurekstraksie is die hoeksteen van verskeie molekulêre opsporingstegnieke, insluitend tradisionele polimerase kettingreaksie (PKR) gebaseerde metodes en hoëdeurset-volgordebepaling (HTS). Die ekstraksiemetode het ‘n noemenswaardige invloed op die tipe, kwaliteit en hoeveelheid RNA wat geı̈soleer word, en gevolglik ook op die algehele opsporingsvermoë van die toets. Soos wat die gewildheid van HTS vir toepassing in hierdie praktyke toeneem, is dit belangrik om verskillende nukleı̈ensuurekstraksie- en volgordebepalingsmetodes te evalueer om sodoende te verseker dat die volkome patogeenopsporingpyplyn geoptimaliseer is. Illumina-platforms het alreeds uitgebreide validering ondergaan in hierdie konteks, maar meer onlangs het Oxford Nanopore Technologies (ONT) ‘n moontlike mededinger geword vir integrasie in hierdie praktyke. Die aanhoudende ontwikkeling van HTS maak ‘n vernuwende vergelyking tussen hierdie twee platforms noodsaaklik. Die doelwit van hierdie studie is om ‘n geskikte RNA-ekstraksiemetode te identifiseer wat die konsekwente, effektiewe en betroubare opsporing van sitrusvirus- en viroı̈ed-RNA met beide tru-transkripsie quantitatiewe PKR (RT-qPKR) en HTS moontlik maak. In totaal is vyf metodes geselekteer: setieltrimetielammoniumbromied (CTAB)/litiumchloried (CL), CTAB/etanol (CE), suur fenolbuffer/litiumchloried/isopropanol (TL), natriumdodeksielsulfaat (SDS)/isopropanol (SI) en CTAB/isopropanol (CP). Die patogeenhoeveelhede in sitrus wat met sitrus tristeza virus (CTV), sitrus virus A (CiVA), sitrus “tatter leaf” virus (CTLV), sitrus dwergviroid (CDVd), sitrus exocortis viroid (CEVd) en “hop stunt” viroid (HSVd) geı̈nfekteer is, is bepaal met RT-qPCR en daarna ook met Illumina HTS opgevolg. Ekstrakte van een metode is geselekteer en gebruik om Illumina en ONT virus- en viroı̈edvolgordebepaling te vergelyk. Die ekstraksiemetode het ‘n merkwaardige invloed gehad op differensiële patogeenopsporing vir beide RT-qPCR en HTS. Die CL-metode het die beste presteer vir CTV, terwyl die SI-metode meer sensitief was vir die ander twee virusse. Viroı̈edopsporing was meer gevarieerd, met CE-ekstrakte wat meer viroı̈ed-RNA bevat het, alhoewel die CL-metode vergelykbare hoeveelhede CDVd gelewer het. Die CE-metode het egter ‘n nadelige effek op virusopsporing gehad, maar die modifikasie aan die metode het dit verbeter, en alhoewel viroı̈edhoeveelhede verminder het, was dit steeds hoog. Ten spyte van die differensiële patogeenekstraksie tussen metodes, kon alle teikenpatogene met die gebruik van geskikte RT-qPCR-replikate en gekombineerde bioinformatiese metodes opgespoor word. Die studie beklemtoon dat alhoewel veelvuldige metodes effektief kan wees, verdere seleksie en optimalisering die prestasie daarvan verder kan verbeter. Die SI-ekstrakte is gekies vir die vergelyking van volgordebepalingsplatforms, en beide tegnologieë kon alle patogene met verwysingsafhanklike en -onafhanklike benaderings opspoor. Illumina het sy gevestigde reputasie van hoë sensitiwiteit, genoomdekking en akkuraatheid gehandhaaf. ONT het vir minder patogeenvolgordefragmente en laer diepte gekompenseer met langer volgordefragmente wat gelei het tot goeie genoomdekking en volgorde-identiteite, vergelykbaar met die wat deur Illumina resultate opgelewer is, mits geskikte vloeiselkapasiteit gebruik is. Die vergelyking tussen Illumina- en ONT-platforms het die sterkpunte wat met elk geassosieer word, beklemtoon en nuwe insigte in die moontlike toepassing van HTS in plantgesondheidsmonitering- en biosekuriteitsprogramme verskaf. Huidige navorsing dien as ‘n fondasie vir die verfyning en verbetering van diagnostiese toetsing in sitruspatologie. Masters 2025-12-18T08:05:14Z 2025-12-18T08:05:14Z 2025-12 Thesis https://scholar.sun.ac.za/handle/10019.1/134602 en Stellenbosch University xviii, 140 pages : illustrations application/pdf Stellenbosch : Stellenbosch University |
| spellingShingle | Citrus -- Diseases and pests Plant viruses -- Detection Viroids RNA -- Detection Polymerase chain reaction UCTD Dippenaar, Madelein Evaluating RNA extraction methods for the detection of citrus viruses and viroids with RT-qPCR and HTS |
| title | Evaluating RNA extraction methods for the detection of citrus viruses and viroids with RT-qPCR and HTS |
| title_full | Evaluating RNA extraction methods for the detection of citrus viruses and viroids with RT-qPCR and HTS |
| title_fullStr | Evaluating RNA extraction methods for the detection of citrus viruses and viroids with RT-qPCR and HTS |
| title_full_unstemmed | Evaluating RNA extraction methods for the detection of citrus viruses and viroids with RT-qPCR and HTS |
| title_short | Evaluating RNA extraction methods for the detection of citrus viruses and viroids with RT-qPCR and HTS |
| title_sort | evaluating rna extraction methods for the detection of citrus viruses and viroids with rt qpcr and hts |
| topic | Citrus -- Diseases and pests Plant viruses -- Detection Viroids RNA -- Detection Polymerase chain reaction UCTD |
| url | https://scholar.sun.ac.za/handle/10019.1/134602 |
| work_keys_str_mv | AT dippenaarmadelein evaluatingrnaextractionmethodsforthedetectionofcitrusvirusesandviroidswithrtqpcrandhts |