微量全血PCR技术鉴定雏鸡及鸡胚性别

Sex Identification with Direct Whole Blood PCR Amplification in Chicklings and Chicken Embryos

为了便于常规PCR技术在鸡性别鉴定上的推广和应用,本研究以成年家鸡全血为模板,应用常规PCR反应缓冲液和Tap DNA聚合酶直接进行PCR扩增,对50μL PCR反应体系中的血样(0.05～4.0μL)和Tap DNA聚合酶的使用量(0.05～1.5μL),以及循环次数(30～40)进行了优化,在此基础上对血样的抗凝剂和保存温度进行了探讨,并对62只1日龄雏鸡、80枚12日龄和80枚16日龄鸡胚的血样直接PCR进行了性别鉴定。结果表明,采集血样时可以使用ACD、肝素或EDTA作为抗凝剂,血样可以在4、-20或-80℃至少保存3个月;使用0.1μL全血就能完成雏鸡和鸡胚性别鉴定,全血PCR鉴定性别与性腺性别的符合度为100%。与常规PCR相比,全血PCR节约了检测成本,提高了检测效率,减少了交叉污染可能。

For facilitation of conventional PCR technology to identify chicken sex,this study took the whole blood of adult chicks as a sample by the conventional PCR reaction mix and Tap DNA polymerase for direct PCR amplification,optimization was conducted in adult chicken whole blood（from 0.05 to 4.0 μL）,Tap DNA polymerase（from 0.05 to 1.5 μL） and cycle number（from 30 to 40） in 50 μL direct PCR amplification system.Anti-coagulant selection and storage temperature of blood sample were evaluated,sex was identified with direct whole blood PCR amplification and compared with gonadal sex in 62 of 1-day-old chicklings,80 of 12-day-old and 80 of 16-day-old chicken embryos,respectively.The results showed that sex identification with direct PCR amplification with 0.1 μL whole blood sample was fully consistent with the gonadal sex,therefore it was accuracy in chicklings and chicken embryos.ACD,heparin or DETA could be adopted as anticoagulant and blood samples could be stored at 4,-20 or-80 ℃ for 3 months at least.Compared with conventional PCR,the direct whole blood PCR could be economic,efficient and low possible of cross-contamination.