Development of a droplet digital PCR assay to detect illicit glucocorticoid administration in bovine

Abstract

Glucocorticoids are often used illegally in food-producing animals for the growth promotion of livestock animals. In accordance to official chemical methods for glucocorticoid detection, an animal is declared as non-compliant when a residue is identified in the sample. Neverthless, growth promoting molecules can often escape identification due to their rapid elimination or due to the use of non-detectable new generation drugs. Therefore, an indirect screening method able to detect the biological effect of long-term administration of low doses of dexamethasone and prednisolone on livestock has been developed to support official methods. As already described, FKBP5 (FKBP prolyl isomerase 5) expression in bovine thymus is regulated by glucocorticoids, and this specific regulation can be exploited in an indirect screening assay. In the present study, male veal calves and young bulls were considered in three different trials in which estradiol, dexamethasone, and prednisolone were administered alone or in combination with Revalor-200 subcutaneous pellets. Thoracic thymus was sampled from all animals and molecular analysis was performed. A duplex droplet digital PCR assay with EvaGreen® was employed to detect the target gene expression using absolute quantification. The developed droplet digital PCR assay was precise, showing intra- and inter-assay mean coefficient of variation values of about 6.16% and 3.17%, respectively. It was also highly specific (100%) with Youden’s index of 76.92% and 53.57% applied to veal calves and young bulls, respectively. The lowest detection limit in which the target gene expression level was kept constant, was 0.05 ng/μl of cDNA with 1 copies/μL and 0.5 copies/μL for target and reference gene, respectively. This study establishes the basis for using a digital PCR-based assay as an efficient test to identify animals illegally treated with glucocorticoids.

A Guide to Biodetection in Droplets

“…Sample partitioning into droplets containing different reactions enhances massive parallelization, which is important for samples limited in volume or containing low concentration of nucleic acids. ,− Divari et al 2022 (Figure ) used ddPCR to study expression genes encoding hormones in livestock animals to monitor the illegal use of supplements. The FKBP5 (FKBP prolyl isomerase 5) gene expression in bovine thymus is regulated by glucocorticoids and thus was used as the target . Commercially available dsDNA binding dye (EvaGreen) was used to label the target (specific amplicons) following fluorescence intensity readouts.…”

“…The FKBP5 (FKBP prolyl isomerase 5) gene expression in bovine thymus is regulated by glucocorticoids and thus was used as the target. 26 Commercially available dsDNA binding dye (EvaGreen) was used to label the target (specific amplicons) following fluorescence intensity readouts. Sensitivity of the assay was estimated at 0.05 ng per μL of cDNA for the FKBP5 target gene and 1 copy per μL of a reference gene.…”