Digital PCR as a new highly sensitive method in chicken cytokine profiling

Chicken cytokine profiling is an established tool used to study the status of the immune system of animals for research and diagnostic purposes. Real-time quantitative PCR (qPCR) is now the simplest and fastest method for gene expression analysis. However, the major disadvantage of this technique is its limited sensitivity in the detection of low expressed genes. Digital PCR (dPCR), on the other hand, is a more recent technique, based on a highly sensitive end point absolute quantification of gene copy number. The aim of this study was to compare cytokine expression in chicken HD-11 cells infected with Salmonella Enteritidis using both qPCR and dPCR. For 6 h, cells were infected with the bacterium alone (Positive Control) or infected and treated with different antibiotics or botanicals, a group without infection and treatments was included (Negative Control; n = 3 all groups). For the qPCR, cDNA was reverse-transcribed and the analysis was performed using PowerUp SYBR Green Master Mix kit. For the dPCR, the QIAcuity OneStep Advanced Probe Kit was used to perform the analysis on RNA samples. The IL1β, IL6, IL10, and IFNγ cytokines was investigated. To compare the results of the 2 methods, data were expressed as 2^(Treatment Ct – Negative Control Ct) for qPCR or as the difference of copies/uL between Treatment and Negative Control for dPCR. Data were analyzed with oneway ANOVA followed by Tukey’s multiple comparisons test. Differences were considered significant at P ≤ 0.05. Statistical significances for IL10 and IFNγ were equivalent comparing the 2 methods. For IL1β, no differences were registered with qPCR, whereas dPCR data indicated statistical significance among groups (P = 0.003). The dPCR provided several significant differences that the qPCR was not able to highlight for IL6. This could be explained by the greater sensitivity of dPCR in detecting copy numbers of IL6 gene in the Negative Control group, because the qPCR was not able to determine the Ct. Also the IFNγ Ct of several samples resulted as undetermined in qPCR analysis, in contrast to dPCR. dPCR can therefore be a candidate technique to replace qPCR in chicken cytokine profiling thanks to its high sensitivity.

Key Words: dPCR, cytokine profiling.

Presented at the 10th Symposium on Gut Health in Production of Food Animals 2022, St. Louis, USA.