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A multiplex bead-based assay for immune gene expression analysis in shrimp

Arayamethakorn, Sopacha, Karoonuthaisiri, Nitsara, Rungrassamee, Wanilada
Journal of biotechnology 2017
DNA primers, Penaeus monodon, Vibrio harveyi, antimicrobial properties, essential genes, feed additives, gene expression, gene expression regulation, gills, lysozyme, melanization, oligonucleotide probes, pathogens, proteins, quantitative polymerase chain reaction, shrimp
Here, we developed a 9-plex bead-based array as a tool to evaluate molecular effects on transcription levels of immune-related genes in the black tiger shrimp (Penaeus monodon). The bead array technology allows simultaneous detection of multiple target genes in a single sample, reducing time, labor and cost. The oligonucleotide probes were designed to target eight immune-related genes that involve in antimicrobial activity, melanization, pathogen pattern recognition proteins, lysozyme and one housekeeping gene as an internal control. The nine probes were coupled to carboxylated-magnetic bead sets. The 9-plex PCR primers were designed and optimized for conditions to allow multiplex detection. The specificity of the assay was validated and the sensitivity was determined to be 103 copies/μL for all target genes. The 9-plex immune gene expression assay was applied to determine transcript levels in gills of P. monodon under exposure to a shrimp pathogen, Vibrio harveyi, and gene expression patterns were consistent to patterns observed under a traditional realtime PCR method. While realtime PCR method gave a better sensitivity but limited multiplexity, our 9-plex immune gene expression assay was able to simultaneously measure expression of multiple target genes, providing useful alternative assay in the need of higher-throughput gene expression analysis such as evaluation of immune stimulatory effects in different feed additives under various dosages and time points in shrimp.