Development and Application of a Screening Method of Absolute Quantitative PCR To Detect the Abuse of Sex Steroid Hormone Administration in Male Bovines

Cucuzza, L. Starvaggi; Biolatti, B.; Divari, Sara; Pregel, Paola; Scaglione, Frine Eleonora; Sereno, Alessandra; Cannizzo, Francesca Tiziana

doi:10.1021/acs.jafc.7b00852

Cited by 7 publications

(3 citation statements)

References 22 publications

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“…FKBP5 gene expression profile detected in this study by ddPCR in thoracic thymus was comparable to previous results obtained using qPCR technology [14], as well as specificity and sensitivity parameters described in analogue qPCR screening tests [23,42].…”

Section: Plos Onesupporting

confidence: 89%

“…Since the output of the analysis is given in copies of cDNA per microliters of reaction (with 95% confidence intervals), ddPCR system allows measurement of target gene expression with high precision calculated with intra-and inter-assay. In fact, the relative CV% values obtained using ddPCR technology were significantly lower than those calculated using qPCR in similar screening methods previously developed [23,42] in which mean intra-and inter-assay CV% were respectively 26.05% and 22.32% [23] and 20.91% and 19.66% [42].…”

Section: Plos Onementioning

confidence: 58%

“…The correlation analysis between duplex and simplex mode is statistically significant allowing to apply the method as duplex form, reducing time and costs of the assay. Moreover, the analysis is faster since ddPCR do not require technical replicates unlike the screening tests previously described [23,43].…”

Section: Plos Onementioning

confidence: 99%

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

et al. 2022

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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.

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Section: Plos Onesupporting

confidence: 89%

Section: Plos Onementioning

confidence: 58%

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

et al. 2022

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Transgenerational expression profiles of a sex related and an epigenetic control gene in the rotifer Brachionus plicatilis in relation to environmental predictability

et al. 2023

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A non-genetic transgenerational inhibitory effect on sexual reproduction has been demonstrated in Brachionus plicatilis in relation to environmental predictability. Indeed, clones of this species from more predictable environments do not respond to sex-inducing cues during several generations after leaving diapause. Notwithstanding, the molecular basis of this effect is still unknown. In this work, the expression level of genes related to the synthesis of sex hormones and to a potential epigenetic signalling mechanism were tracked along successive generations from diapausing eggs in clones of B. plicatilis populations inhabiting ponds with different level of environmental predictability. The selected genes were (1) the 17-β-dehydrogenase gene (edh), involved in the synthesis of 17-β-estradiol hormone in rotifers, and (2) the DNMT2 gene (meth), as a candidate epigenetic mechanism of control. According to expectations, results showed an increasing expression of edh across generations in clones from those the more predictable ponds. This finding provides a putative role of estradiol in the transgenerational effect. However, no differences were found in the meth gene neither across generations nor regarding the environmental predictability. Despite this, we point out alternatives for future research on the inherited gene regulation mechanism behind the transgenerational effect.

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Application of RNA-sequencing to identify biomarkers in broiler chickens prophylactic administered with antimicrobial agents

Giannuzzi

Biolatti

Longato

et al. 2021

animal

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Development and Application of a Screening Method of Absolute Quantitative PCR To Detect the Abuse of Sex Steroid Hormone Administration in Male Bovines

Cited by 7 publications

References 22 publications

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

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

Transgenerational expression profiles of a sex related and an epigenetic control gene in the rotifer Brachionus plicatilis in relation to environmental predictability

Application of RNA-sequencing to identify biomarkers in broiler chickens prophylactic administered with antimicrobial agents

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