Rapid Single Cell Typing using SYBR® Green Real-Time PCR Together with Melt Curve Analysis for Sex Identification of Porcine Sperm

Abstract

Identification of X or Y chromosome is a very useful technique to verify the sex of boar sperm, but common methods used in pig such as Fluorescence In situ Hybridisation (FISH) and whole semen Polymerase Chain Reaction (PCR) have some limitations. FISH is highly accurate, but time-consuming (>3 days). Whole semen PCR is faster than FISH (3-6 h), but not highly accurate (approximate methods). The objective of this study was to develop a fast and highly accurate protocol to identify sex of boar sperm. In the present study, our team developed an alternative sex identification protocol using single cell SYBR® green real-time PCR technique together with low resolution melt curve analysis. Primers specific for chromosome 1 and chromosome Y, a high performance KAPA SYBR® DNA polymerase and Rotor gene PCR platform were used. Male and female single white blood cells were used to calculate sensitivity and specificity. Single sperm was picked up under inverted microscope and transferred to 1 μl of lysis buffer, and real-time PCR was run according to the programmed protocol and analyzed with melt curve analysis. Results showed that our method was a fast (<50 min) accurate method with high sensitivity (95-99%) and specificity (100%) with low percentage of PCR failure (< 3%). Validation of this method using boar whole semen detected Y sperm at 52% and X sperm at 48%, which was comparable to the theory ratio of X and Y sperm (50:50) in semen. It may be concluded that the single cell SYBR® green real-time PCR technique together with melt curve analysis is fast and accurate that can be used to identify sex of boar sperm.