猪性控精子纯度实时荧光定量PCR检测方法的建立

doi:10.16431/j.cnki.1671-7236.2021.10.019

Abstract

Abstract: The aim of this study was to use Real-time quantitative PCR technique to detect sexually controlled sperm of Duroc pigs, in order to establish a rapid, accurate and cost-effective method for sperm purity detection. The sex -determining region Y (SRY) gene located on the Y chromosome and A-kinase anchoring protein 4 (AKAP4) gene located on the X chromosome of pig were selected, using genomic DNA extracted from pig ear tissue samples as a template for PCR amplification for specific primer verification. The gel recovery kit was used to recover the gel, the plasmid were extracted. After testing, the obtained two plasmids were diluted to the same concentration (20 ng/μL), and mixed to construct plasmid templates containing different proportions of SRY and AKAP4 genes, which were used to construct the reaction template for the standard curve of sperm purity detection. The purity of 3 mixed X spermatozoa (P. x_gro1, P. x_gro2 and P. x_gro3) and 3 mixed Y spermatozoa (P. y_gro1, P. y_gro2 and P. y_gro3) were detected by SRY and AKAP4 genes specific primers, respectively. The results showed that the purity obtained using SRY primers of P. x_gro1, P. x_gro2 and P. x_gro3 sperm groups were 91.44%, 91.93% and 88.99%, and the purity of P. y_gro1, P. y_gro2 and P. y_gro3 sperm groups were 89.91%, 87.31% and 88.71%, respectively. The purity obtained by using AKAP4 primers of P. x_gro1, P. x_gro2, P. x_gro3 sperm groups were 91.44%, 91.93% and 88.99%, and the purity of P. y_gro1, P. y_gro2, P. y_gro3 sperm groups were 89.91%, 87.31% and 88.71%, respectively. Independent sample t-test analysis showed that no significant difference between the two test results, indicating that the results were accurate for sperm purity detection. In this experiment, Real-time quantitative PCR was used to accurately detect the purity of sperm sorted by flow cytometry, and established a new method for detecting the ratio of X sperm and Y sperm in porcine sperm samples by Real-time quantitative PCR.

Key words: pig; sex-controlled sperm; purity test; standard curve; Real-time quantitative PCR

CLC Number:

S828

LI Zhiqiang, TAO Chenyu, WEI Aolong, JIA Qing. Establish on Real-time Quantitative PCR Method for Purity Detection of Porcine Sex-controlled Sperm[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(10): 3682-3690.

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Establish on Real-time Quantitative PCR Method for Purity Detection of Porcine Sex-controlled Sperm

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