Abstract: Objective　To establish a method for genotyping of NOD/ShiLt mice.Methods　For the deletion sequence on chromosome 3 of NOD/ShiLt strain and reference gene GAPDH, specific primers were designed. Standard plasmids were constructed to establish a SYBR Green fluorescence quantitative PCR(qPCR) method for the detection of NOD/ShiLt. After its sensitivity, specificity and repeatability were verified, preserved NOD/ShiLt mouse sperm and embryo samples were examined.Results　The detection limit of the established qPCR was 6.35×100 copies/ μL, 1 000-fold higher than that of normal PCR. No cross-reactions were found with other commonly used strains including background strains ICR. The coefficients of variation within and between batches were 0.05%-0.67% and 0.07%-0.77%, respectively, both lower than 1.00%, indicating good repeatability. Frozen NOD/ShiLt and NPI embryos and spermatozoa were detected by this method and the coincidence rate was 100%.Conclusion　This study successfully established a highly sensitive detection method for NOD/ShiLt strains, which can be used for genotype detection of NOD/ShiLt samples including sperm and embryo.

Key words: NOD, sperm, embryo, genotype identification, fluorescence quantitative PCR 

摘要： 目的　建立适用于NOD/ShiLt小鼠基因型鉴定的荧光定量PCR方法。方法　针对NOD/ShiLt品系3号染色 体上的缺失序列和内参基因GAPDH设计特异性引物,优化建立检测 NOD/ShiLt小鼠基因型的SYBR Green荧光定 量PCR(qPCR)方法,在验证其敏感性、特异性和可重复性后,对保存的NOD/ShiLt小鼠精子和胚胎样本进行检测。 结果　所建荧光PCR检测限为6.35×100拷贝/μL,高于普通PCR 1 000倍;检测其他常用品系包括背景品系ICR, 特异性良好;批内、批间变异系数分别为0.05%~0.67%和0.07%~0.77%,均低于1.00%,重复性良好。利用该方 法对冻存的NOD/ShiLt和NPI品系胚胎及精子进行检测,符合率100%。结论　本实验成功建立了NOD/ShiLt品 系的高敏感检测方法,可用于NOD/ShiLt包括精子、胚胎等样本在内的基因型检测。

关键词: NOD, 精子, 胚胎, 基因型鉴定, 荧光定量PCR