Developing a Nested Real-Time PCR Assay for Determining Equine Fetal Sex Prenatally

• In this study, fetal sex determined by analyzing cell-free fetal DNA after 8 weeks of gestation.
• Maternal plasma was used for analysis.
• The technique was based on real-time PCR.

The aim of this investigation was to develop a real-time polymerase chain reaction (PCR) assay for detecting circulating cell-free fetal DNA in Arabian pregnant mare plasma and predicting fetal sex through identifying SRY gene. Peripheral blood samples of 28 pregnant mares were collected into tubes containing EDTA, at a single time point, between 8 and 20 weeks of gestation. Circulating cell-free fetal DNA was extracted from 3 mL of maternal plasma. Using outer and inner primers, real-time PCR was performed for the SRY gene and for the glyceraldehyde-3-phosphate dehydrogenase gene as the internal control. Amplicons were labeled as positive, negative, or inconclusive according to threshold cycle and melting curve analysis. Of the total 28 Arabian pregnant mares, 16 were carrying male and 12 were carrying female fetuses. On first round of real-time PCR, results were inconclusive for SRY gene; however, on the second round, conclusive results were obtained. In total, the accuracy of test was 88% (22/25) (confidence interval [CI]: 65.5, 95.6); it had a sensitivity of 85.7% (CI: 65.6, 92.5), whereas its specificity was 90.9% (CI: 65.3, 99.5). We had a positive predictive value of 92.3% (CI: 70.7, 99.6) and a negative predictive value of 83.3% (CI: 59.9, 91.2). To conclude, SRY gene can permit detection of equine fetal sex with a good accuracy by analyzing cell-free fetal DNA in maternal plasma just after 8 weeks of gestation.