Selection of highly specific aptamers to detect
Fusarium graminearum and Fusarium
pseudograminearum
A thesis submitted in fulfilment of the requirements for the degree of Master of Science
Sanskruti Lakhotia
M.Sc., Madurai Kamaraj University
School of Science
College of Science, Engineering and Health
RMIT University
June 2019
Declaration
I certify that except where due acknowledgement has been made, the work is that of the
author alone; the work has not been submitted previously, in whole or in part, to qualify
for any other academic award; the content of the thesis is the result of work which has
been carried out since the official commencement date of the approved research program;
any editorial work, paid or unpaid, carried out by a third party is acknowledged; and, ethics
procedures and guidelines have been followed.
Sanskruti Lakhotia
28 June 2019
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Abstract
The aim of this project was to select highly sensitive and specific aptamer capable of
detecting Fusarium graminearum (Fg) and F. pseudograminearum (Fp) species. The
aptamer-based testing allows early detection of fungal infection in the field. Fungi are
known to change their activity and may often undergo genetic changes. The classic
“gene for gene” theory explains the relationship between host plant cultivars and
fungal pathogenic races. To avoid the induction of resistance in plant cultivars, fungi
changes function of some virulence genes, thus giving rise to new pathogenic strains.
Loss of pathogenicity during culture storage is frequently observed in fungi. This could
be due to spontaneous mutations or segregation of heterokaryotic or
extrachromosomal elements. For these reasons, we isolated fusarium strains from
field grown wheat plants that exhibited fusarium-specific symptoms. The stem sections
from infected plants were sub-cultured on potato dextrose agar and pure cultures were
obtained by serial dilution and spread plating. Subsequently, molecular identification
of fusarium species was performed using species-specific primers. Further, to confirm
the species of the fungus and the purity of the culture, ITS6 region was sequenced.
The BLAST results of sequencing data confirmed isolation of pure cultures of Fg and
Fp from filed samples.
Biosensors are attractive for field testing because they are cost-effective, portable and
can be used even by a lay person. Sensors used currently in the agriculture pathogen
detection market are based on antibodies, which have low stability and their production
is difficult as it involves use of animals. An alternative for antibodies are DNA/RNA
aptamers that can be chemically synthesized at a low cost and are thermally stable.
Aptamers bind to their target molecules with high affinity and specificity through
complementary spatial configuration. Aptamers can bind to proteins, enzymes,
biotoxins, or even whole cells. We utilised the repetitive selection-amplification
process known as systematic evolution of ligands by exponential enrichment (SELEX)
for selection of aptamers specific to Fp and Fg spores. The SELEX process required
no prior knowledge of cell surface molecules on the target cell and allows detection of
whole live cell.
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The cell SELEX was performed using a commercially available N40 aptamer library
with known forward and reverse flanking sequences on the sides of random single-
stranded 40 bp DNA sequences (Trilink DNA technologies). Fluorescence Activated
Cell Sorting was used to select aptamers binding to fungal spores. The bound
aptamers were amplified thrice. Negative selection was used to eliminate those
aptamers that might bind to other fungal species infecting wheat. After 3 cycles of
positive selection, alternating cycles of negative selection was performed for a total of
9 cycles. Subsequently, the pool obtained was cloned into pCR XL-2-TOPO vector,
transformed to E. coli and sequenced. On analysing the sequencing data, 43 and 31
different aptamers binding to Fg and Fp, respectively were identified. These aptamers
were analysed using in silico and phylogenetic analysis. Four aptamers with the
maximum number of copies were selected from both pools. Out of these, two had high
affinity towards both the fusarium species (also present in maximum copies) and two
were specific to each of the species. After identifying these four optimal aptamers,
these were commercially synthesized and the specificity was confirmed using confocal
microscopy and scanning electron microscopy (SEM). In addition, the dissociation
constants (Kd) of these four aptamers was determined to select the best one for
generating a nano-aptasensor.
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Dedicated to Mummy, Papa and Swayam