Development and validation of a qPCR assay for the non-invasive determination of fetal sex in cattle and African Buffalo

Abstract

The expansive possibilities and research progress of cell-free DNA (cfDNA) and cell-free foetal DNA (cffDNA) have engendered numerous articles published on a regular basis in recent years. However, this has mainly been centred on early diagnostic protocols and detection of different types of cancer or non-invasive genetic testing of human fetuses. Various challenges and novel aspects still exist regarding cfDNA and cffDNA when it comes to investigating human samples. Work on the animal side of the spectrum has been scarce and most studies have focused on mouse models to be used for human testing at later stages. Knowledge regarding the presence of cfDNA and cffDNA in cattle and African buffalo biological fluids is substantially insufficient. This study attempted to be the first to confirm the presence of cffDNA in maternal plasma of African buffalo. The study used cffDNA isolated from cattle and African buffalo plasma samples to determine the foetal sex of the animals non-invasively. The aim of the study was to develop a practical and robust method that could be used on a daily basis by veterinarians. cfDNA was isolated from cattle and African buffalo plasma samples, confirming the presence of cffDNA in the maternal plasma of these animals. We attempted to amplify the isolated cfDNA by means of real-time PCR (qPCR) to confirm the presence of the sex determining region y (SRY) gene only present in male animals. After qPCR of the maternal plasma samples the results were compared to the sex of the calves after birth. Because of the contamination of the nuclease-free water that occurred during the qPCR step of the cattle samples, 50% of samples presented inconclusive results in both of the duplicates, while 27% of the results were correct in both duplicates and 23% of the samples amplified incorrectly. The contamination was eliminated during the qPCR step of the African buffalo duplicates. A total of 44% of samples were correct in both replicates, but only one of the nine male samples amplified in both replicates. A further 19% of male samples amplified in at least one of the replicates with no false positives present. Based on the high number of false negatives considerable work remains to improve these methods for future studies. This might include alternate and more sensitive methods to qPCR and more specialised isolation procedures.