Thesis (MSc)--University of Stellenbosch, 2008. / ENGLISH ABSTRACT: Potato virus Y (PVY) is responsible for considerable yield losses in the South African potato industry.
The incidence of this virus has greatly increased over the past few years. Even more worrying is the
variation of symptoms observed during PVY infection and the recent appearance of the more virulent
PVYNTN strain in local fields. This project aimed to investigate the possible genetic variation within the
viral genome and to establish the origin of strains. The project also aimed to establish a dependable, area
specific enzyme-linked immunosorbent assay (ELISA) to replace the currently used ELISAs. Currently
seed potato certification is done using ELISA kits imported from Europe. These kits were developed for
the detection of overseas variants of PVY and the use thereof in South Africa has in the past lead to false
negatives. Finally, this project set out to develop, optimize and establish a sensitive and reliable real-time
reverse transcriptase polymerase chain reaction (qRT-PCR) detection protocol for PVY.
In the first part of the study the coat protein (CP) gene of PVY isolates from plant material obtained from
various parts of South Africa was amplified using RT-PCR. The resulting cDNA was then sequenced
directly or cloned into a vector and then sequenced. The resulting sequences were aligned in a data matrix
with international reference sequences, analyzed and grouped according to strain. Examination of the CP
gene within this matrix as well as phylogenetic analysis revealed six main groups of PVY. These six
groups included the traditional PVYN and PVYO groups and a recombinant group. Furthermore it also
revealed variants of PVYN and PVYO. These mutants and recombinants pose a threat as they may lead to
South African strains of PVY expressing coat proteins which vary from those found overseas. This may
render the currently used European ELISA method of detection less effective and subsequently result in
an increase in viral prevalence. This reinforced the need for a detection method based on local viral
strains. Phylogenetic and Simplot analysis also confirmed that a recombinant strain between PVYN and
PVYO had evolved and that PVYNTN was such a recombinant.
The second part of the study aimed to develop and establish detection methods based on local variants of
PVY. This included the development of ELISA and qRT-PCR detection methods of PVY. Previously
amplified cDNA of the PVY CP gene was cloned into an expression vector and successfully expressed.
Antibodies produced against the recombinant protein, when used in ELISA, however, failed to achieve
the required levels of sensitivity. This prompted the development of qRT-PCR detection methods for
PVY. Primer combinations for PVY were designed using the previously established CP gene data matrix.
A reliable and sensitive SYBR® Green I based qRT-PCR assay was developed for the detection of PVY.
The assay effectively detected all known South African variants of PVY. Furthermore, a Taqman® assay
was developed for the detection of all variants of PVY. The Taqman® assay was 10 fold less sensitive and
does not allow for amplicon verification through melting curve analysis, but it does add more specificity
due to the addition of the probe. Although these qRT-PCR detection methods are still too expensive to
replace the routine diagnostics done with ELISA, they do offer the opportunity to screen valuable mother
material and confirm borderline cases in seed certification. / AFRIKAANSE OPSOMMING: Aartappel virus Y (PVY) is verantwoordelik vir aansienlike opbrengsverliese in die Suid-Afrikaanse
aartappelindustrie. Die insidensie van infeksie deur die virus het drasties toegeneem oor die afgelope jare.
Wat egter meer kommerwekkend is, is die groter variasie in simptome van PVY infeksie en die onlangse
voorkoms ‘n meer virulente ras, PVYNTN. Hierdie projek poog om moontlike genetiese variasie van PVY
te ondersoek en om die oorsprong van rasse op te spoor. Die projek het ook gepoog ook om ‘n bruikbare,
betroubare en area spesifieke “enzyme-linked immunosorbent assay” (ELISA) toets te ontwikkel om die
huidige ingevoerde ELISA te vervang. Hierdie toetse is ontwikkel om oorsese variante van PVY op te
spoor en die gebruik daarvan het in die verlede gelei tot vals negatiewes. Verder is daar ook ondersoek
ingestel na die ontwikkeling van ‘n sensitiewe en betroubare “real-time reverse transcriptase polymerase
chain reaction” (qRT-PCR) protokol vir die opsporing van PVY.
In die eerste deel van die studie is die mantelproteïen geen van PVY isolate vanuit plant materiaal
geamplifiseer deur die gebruik van RT-PCR. Hierdie materiaal is vanaf verskeie streke in Suid-Afrika
ontvang. ‘n Volgordebepalingsreaksie is uitgevoer op gekloneerde of ongekloneerde cDNA verkry uit die
RT-PCR. DNA volgordes is in ‘n data matriks geplaas en vergelyk met internationale volgordes om die
plaaslike isolate te analiseer en te groepeer. Deur vergelyking en filogenetiese ontleding kon ses
hoofgroepe van PVY geïdentifiseer word, wat tradisionele PVYN en PVYO, sowel as ‘n rekombinante ras
en variante binne die tradisionele PVYN en PVYO groepe ingesluit het. Rekombinante en mutante kan
veroorsaak dat Suid-Afrikanse rasse van PVY mantelproteïene uitdruk wat afwyk van die oorsese rasse
wat tot gevolg mag hê dat die ELISAs van oorsee minder effektief kan wees en kan lei tot verhoogde
virus voorkoms. Die realiteit en gevaar versterk die gedagte dat ‘n deteksie metode gebaseer op plaaslike
virusse absoluut krities is. Filogenetiese sowel as Simplot analise het bevestig dat ’n mutante ras tussen
PVYN en PVYO ontstaan het en dat PVYNTN ’n rekombinante ras is.
Die tweede deel van die studie was daarop gemik om deteksie metodes te ontwikkel wat gebaseer was op
plaaslike variante van PVY. Dit sluit die ontwikkeling van ELISA sowel as qRT-PCR deteksie van PVY
in. Voorheen geamplifiseerde cDNA is in ‘n ekspressievektor gekloneer en suksesvol uitgedruk.
Teenliggaampies teen die rekombinante proteïen, indien in ELISA aangewend, kon egter nie die nodige
sensitiwiteit oplewer nie. Dit het aanleiding gegee tot ontwikkeling van qRT-PCR deteksie metodes.
Inleier kombinasies vir PVY was ontwikkel deur die gebruik van die bestaande mantelproteïen geen data
matrikse. ‘n Betroubare en sensitiewe SYBR® Green I qRT-PCR deteksie protokol was ontwikkel vir die
effektiewe deteksie van alle bekende Suid-Afrikanse rasse van PVY. Verder is ‘n sogenaamde
“Taqman®” protokol ook ontwikkel vir deteksie van alle rasse. Die “Taqman®” protokol was 10 voudiglik
minder gevoelig and laat nie bevestiging deur smeltkurwe analise toe nie, maar verleen meer spesifisiteit
deur die toevoeging van die “Taqman® probe”. Hierdie qRT-PCR deteksie metodes is tans te duur om as
roetine diagnostiese toetse te gebruik en kan dus nie ELISA vervang nie, maar hulle bied wel die
geleentheid om waardevolle moeder materiaal te toets en grensgevalle in aartappelsaad sertifisering te
bevestig.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/21878 |
| Date | 03 1900 |
| Creators | Visser, Johan Christiaan |
| Contributors | Bellstedt, D.U., Stellenbosch University. Faculty of Science. Dept. of Biochemistry. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | Unknown |
| Type | Thesis |
| Format | 78 leaves : ill. |
| Rights | Stellenbosch University |
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