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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.