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Development of a CRISPR-based 'Candidatus Liberibacter africanus' detection system
Thesis (MScAgric)--Stellenbosch University, 2024.
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| Format: | Thesis |
| Language: | English |
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Stellenbosch : Stellenbosch University
2025
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| _version_ | 1867613802460086272 |
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| access_status_str | Open Access |
| author | Dove, Joshua Charles |
| author2 | Burger, Johan |
| author_browse | Burger, Johan Dove, Joshua Charles |
| author_facet | Burger, Johan Dove, Joshua Charles |
| author_sort | Dove, Joshua Charles |
| collection | Thesis |
| dc_rights_str_mv | Stellenbosch University |
| description | Thesis (MScAgric)--Stellenbosch University, 2024. |
| format | Thesis |
| id | oai:scholar.sun.ac.za:10019.1/131646 |
| institution | Stellenbosch University (South Africa) |
| language | English |
| last_indexed | 2026-06-10T12:41:56.100Z |
| 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:10019.1/131646 Development of a CRISPR-based 'Candidatus Liberibacter africanus' detection system Dove, Joshua Charles Burger, Johan Campa, Manuela Stellenbosch University. Faculty of Agrisciences. Dept. of Genetics. Greening disease -- South Africa Citrus fruit industry -- South Africa CRISPR (Genetics) Citrus -- Breeding RNA Candidatus Liberibacter africanus Citrus -- Diseases and pests UCTD Thesis (MScAgric)--Stellenbosch University, 2024. ENGLISH ABSTRACT: Citrus greening (Huanglongbing or HLB) poses a substantial threat to global citrus industries, causing severe economic losses and declining productivity. In regions like South Africa, where the citrus industry is a significant economic contributor, HLB could be particularly threatening. This project addresses the need for effective detection systems to mitigate the devastating effects of HLB on citrus cultivation. Despite the severity of HLB, there is currently no cure, emphasising the crucial role of rapid and effective diagnostic tools for timely identification and disease management. Current detection methodologies like PCR and qPCR, while effective, face limitations in terms of cost, requirement of skilled labour, sophisticated equipment and slow turnaround times. Recently, CRISPR-based diagnostics have been implemented as a means of detection for a wide variety of bacterial, fungal and viral pathogens. These systems show promise in terms of cost-effectiveness, ease of use, sensitivity and turnaround time. To this end, this study introduces a proof-of-concept detection system focusing on ‘Candidatus Liberibacter africanus’ (CLaf), specifically targeting the ribonucleotide reductase (RNR) gene of the bacterium using the collateral cleavage abilities of CRISPR/Cas12a for nucleic acid detection. To enhance sensitivity, the CRISPR/Cas12a detection method is integrated with recombinase polymerase amplification (RPA), providing rapid and efficient DNA preamplification. The proposed diagnostic system aligns with practical agricultural considerations, streamlining the detection process while effectively addressing technical challenges associated with low CLaf titres in plant tissues. This study explores various aspects of the RPA- Cas12a assay, starting with nucleic acid extraction methods. While successful extractions were achieved, challenges in detecting CLaf in the insect vector, Trioza erytreae, highlight the need for optimisation in CRISPR assay conditions, target regions, or alternative methods for enhanced sensitivity. Comparing preamplification methods, RPA demonstrated superiority over Loop- Mediated Isothermal Amplification (LAMP), particularly in compatibility with Cas12a's operating temperature. The RPA-Cas12a diagnostic system employs a guide RNA (gRNA) complementary to the target sequence, with Cas12a triggering collateral cleavage of single-stranded DNA fluorescent reporter molecules upon target detection. The development of fluorescence is measured using a Rotor-Gene® Q machine. Further modifications could enable detection using lateral flow strips, contributing to advancing detection technologies for efficient disease management on-site. Achieving a detection sensitivity of 100 aM using spiked crude extracts, this study underscores the RPA-Cas12a system's impressive sensitivity. In analysing 28 citrus plant samples, the assay generally aligned with conventional qPCR diagnostic outcomes. However, instances of discrepancies highlight potential higher sensitivity in detecting low CLaf DNA concentrations compared to qPCR. While facing limitations in psyllid testing and in-field application, the RPA-Cas12a assay demonstrates promise for point-of-care applications in 'Candidatus Liberibacter spp.' detection, particularly in South African citrus orchards. AFRIKAANSE OPSOMMING: Sitrusvergroening (Huanglongbing of HLB) hou 'n wesenlike bedreiging in vir globale sitrusbedrywe, wat ernstige ekonomiese verliese en dalende produktiwiteit veroorsaak. In streke soos Suid-Afrika, waar die sitrusbedryf 'n beduidende ekonomiese bydraer is, kan HLB veral bedreigend wees. Hierdie projek spreek die behoefte aan effektiewe opsporingstelsels aan om die vernietigende uitwerking van HLB op sitrusverbouing te versag. Ten spyte van die erns van HLB, is daar tans geen beheermaatreëls nie, wat dus die deurslaggewende rol van vinnige en effektiewe diagnostiese toetse vir tydige identifikasie en siektebestuur beklemtoon. Huidige opsporingsmetodologieë soos PCR en qPCR, hoewel effektief, ondervind beperkings in terme van koste, die vereiste van geskoolde arbeid, gesofistikeerde toerusting en stadige omkeertye. Onlangs is CRISPR-gebaseerde diagnostiek geïmplementeer as 'n manier van opsporing vir 'n wye verskeidenheid van bakteriële, swam en virale patogene. Hierdie stelsels toon belofte in terme van koste-effektiwiteit, gebruiksgemak, sensitiwiteit en omkeertyd. Vir hierdie doel stel hierdie studie 'n konsep-opsporingstelsel bekend wat fokus op 'Candidatus Liberibacter africanus' (CLaf), wat spesifiek die ribonukleotied reduktase (RNR) geen van die bakterie teiken deur die kollaterale splytsingsvermoëns van CRISPR/Cas12a vir nukleïensure te gebruik. Om sensitiwiteit te verbeter, word die CRISPR/Cas12a-opsporingsmetode geïntegreer met rekombinase-polimerase-amplifikasie (RPA), wat vinnige en doeltreffende DNA-voorversterking verskaf. Die voorgestelde diagnostiese stelsel strook met praktiese landbou-oorwegings, wat die opsporingsproses vaartbelyn maak terwyl tegniese uitdagings wat met lae CLaf-titers in plantweefsels geassosieer word, effektief aangespreek word. Hierdie studie ondersoek verskeie aspekte van die RPA- Cas12a-toets, beginende met nukleïensuur-ekstraksiemetodes. Terwyl suksesvolle ekstraksies gedoen kon word, beklemtoon uitdagings in die opsporing van CLaf in die insekvektor, Trioza erytreae, die behoefte aan optimisering in CRISPR-toetstoestande, teikenstreke of alternatiewe metodes vir verhoogde sensitiwiteit. Deur voorversterkingsmetodes te vergelyk, het RPA beter vertoon as Lus- bemiddelde Isotermiese Amplifikasie (LAMP), veral in terme van verenigbaarheid met Cas12a se operasionele temperatuur. Die RPA-Cas12a-diagnostiese stelsel gebruik 'n gids-RNA (gRNA) wat komplementêr tot die teikenvolgorde is, met Cas12a wat kollaterale splitsing van enkelstring-DNS- fluoresserende verslaggewer-molekule by teikenopsporing veroorsaak. Die ontwikkeling van fluoressensie word gemeet met 'n Rotor-Gene® Q-masjien. Verdere wysigings kan opsporing moontlik maak met behulp van laterale vloeistroke, wat bydra tot die bevordering van opsporingstegnologie vir doeltreffende siektebestuur in die veld. 'n Opsporingsensitiwiteit van 100 aM is bereik deur gebruik te maak van ru-ekstrakte, wat die indrukwekkende sensitiwiteit van die RPA-Cas12a-stelsel beklemtoon. In die toets van 28 sitrusplantmonsters het die toets oor die algemeen goed belyn met konvensionele qPCR-diagnostiese toetse. Gevalle van verskille beklemtoon egter potensiële hoër sensitiwiteit in die opsporing van lae CLaf DNA-konsentrasies in vergelyking met qPCR. Terwyl die RPA-Cas12a-toets beperkings in psyllid- toetsing en in-veld-toepassing in die gesig staar, toon die belofte vir punt-van-sorg toepassings in 'Candidatus Liberibacter spp.' opsporing, veral in Suid-Afrikaanse sitrusboorde. Masters 2025-02-03T10:17:39Z 2025-02-03T10:17:39Z 2024-12 Thesis https://scholar.sun.ac.za/handle/10019.1/131646 en Stellenbosch University ix, 65 pages : illustrations application/pdf Stellenbosch : Stellenbosch University |
| spellingShingle | Greening disease -- South Africa Citrus fruit industry -- South Africa CRISPR (Genetics) Citrus -- Breeding RNA Candidatus Liberibacter africanus Citrus -- Diseases and pests UCTD Dove, Joshua Charles Development of a CRISPR-based 'Candidatus Liberibacter africanus' detection system |
| title | Development of a CRISPR-based 'Candidatus Liberibacter africanus' detection system |
| title_full | Development of a CRISPR-based 'Candidatus Liberibacter africanus' detection system |
| title_fullStr | Development of a CRISPR-based 'Candidatus Liberibacter africanus' detection system |
| title_full_unstemmed | Development of a CRISPR-based 'Candidatus Liberibacter africanus' detection system |
| title_short | Development of a CRISPR-based 'Candidatus Liberibacter africanus' detection system |
| title_sort | development of a crispr based candidatus liberibacter africanus detection system |
| topic | Greening disease -- South Africa Citrus fruit industry -- South Africa CRISPR (Genetics) Citrus -- Breeding RNA Candidatus Liberibacter africanus Citrus -- Diseases and pests UCTD |
| url | https://scholar.sun.ac.za/handle/10019.1/131646 |
| work_keys_str_mv | AT dovejoshuacharles developmentofacrisprbasedcandidatusliberibacterafricanusdetectionsystem |