Development and validation of a SYBR Green real-time PCR assay for rapid and quantitative detection of goose interferons and proinflammatory cytokines

摘要

Real time quantitative polymerase chain reaction (RT-qPCR) based on SYBR-Green I binding is a quick, reliable, and easy method for analyzing small amounts of mRNA. Viral pathogens are recognized at the time of infection by pattern recognition receptors; thus, the inflammatory cytokines (IL1 beta, IL6, and IL18) and antiviral cytokines (IFN alpha, IFN gamma) are secreted by innate immune cells and induced to respond to the pathogens. The objective of this study was to develop an effective and sensitive RT-qPCR assay for the rapid and accurate quantification of goose cytokines: IFN alpha, IFN gamma, IL1 beta, IL6, and IL18. Subsequently, the established methods were employed to detect the immune response in agonist-stimulated goose spleen cells in vitro. These data indicated that the established RT-qPCR is a reliable method for determining relative gene expression. The results revealed that Imiquimod led to the significant upregulation of goose IFN alpha (P 0.01), IFN gamma (P 0.01), IL1 beta (P 0.01), IL6 (P 0.01), and IL18 (P 0.05). The established methods are important for scientific research and clinical applications, which require rapid and accurate results in a short period of time. The technique can potentially be used in the further research of goose molecular immunology, which will help us understand the interactions between hosts and pathogens.