High-throughput OpenArray™: novel, swift TaqMan® Real-Time PCR chip for diagnostic biomarkers in Oncorhynchus tshawytscha
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Date
2024-01-26
Authors
Dang, Xiaoqing
Journal Title
Journal ISSN
Volume Title
Publisher
University of Waterloo
Abstract
In 2014, aquaculture replaced capture fisheries as the primary source of fish supply
for human consumption for the first time in history, but to date, fish domestication has not
developed enough to cope with the disease-related challenges. Members of the Salmonidae
family are among the most popular commercialized fish species and salmon aquaculture has
been the fastest growing food production system worldwide. An OpenArray™ device for
Chinook Salmon (Oncorhynchus tshawytscha) health surveillance was validated in this
thesis. On each microscope slide–sized OpenArray™ plate, 28 gene transcripts from 48
samples can be quantified using TaqMan® RT-qPCR. To evaluate the function of the chip,
fish culture water, mucus, head kidney, spleen, and gill tissues were collected from Chinook
salmon that were intraperitoneally injected with live Vibrio anguillarum within the course of
96 h. Based on the on-chip primer efficiency results, primers for 27 of the genes were
considered acceptable for future validation. Based on the transcript expression patterns
revealed by the OpenArray™ TaqMan® RT-qPCR chip, 6 immune genes (calm, mhc-i, mhc-
ii, il-1β, il-8, tapbp) were selected and validated by SYBR RT-qPCR. Pearson’s correlation
analysis revealed that a moderate to strong correlation existed between the results generated
by both RT-qPCR methods for the 6 genes analysed.
Both OpenArray™ TaqMan® and SYBR RT-qPCR detected significantly increased il-
1β and il-8 expression levels in spleen tissue at 72 h and 96 h, although the peaks of the
expression were not recorded within the time point analysed. In head kidney tissue, il-1β
expression level was also increased at 72 h, but there was no difference observed for il-8
between the control and infected groups within any time points. A longer response initiation
time was observed in the Chinook salmon model in this trial. Comparing to previous studies
in related salmon species, the pro-inflammatory cytokine il-1β was observed to increase as
early as 6 h post infection in vivo. Nevertheless, mucus samples showed great potential as a
minimally invasive sampling method if the transcripts were quantified by SYBR RT-qPCR.
By comparison, in mucus the OpenArray™ TaqMan® RT-qPCR chip did not report as many
quantification data as in spleen and head kidney tissue. In general, both RT-qPCR methods
revealed an early systematic immune response induced by Vibrio anguillarum in spleen and
head kidney, but the biological significance of the delayed immune response initiation will
require further analysis to understand.
The OpenArray™ TaqMan® RT-qPCR chip validated in this thesis was designed to
be specific to salmon species under the order Salmoniformes. By validating this health
monitoring tool in other salmon species, it is possible that the interspecies physiological
differences can be horizontally compared. This will enable the development of species-
specific, more effective prophylactic and therapeutic approaches against the salmon diseases.
Description
Keywords
salmon, transcriptomics, TaqMan RT-qPCR, OpenArray chip, Innate immunology