FISH联用激光捕获显微分离技术对混合斑进行分离检验

Isolation and DNA profiling of spermatozoa from mixed stain by fluorescence in situ hybridization and Laser capture microdissection

目的建立荧光原位杂交(FISH)联用激光捕获显微分离技术(LCM)精确分离男女混合斑中精子的检验方法。方法收集健康志愿者精子和女性阴道上皮细胞制备模拟混合斑,经过预处理后用Vysis CEPX SpectrumOrangeTMY SpectrumGreenTM试剂盒进行荧光原位杂交,并用PALM激光捕获显微分离系统分离男、女性细胞,使用Identifiler试剂盒结合低体积扩增技术分别对男女成分进行STR扩增。结果荧光原位杂交后,可清晰分辨混合斑中的男女细胞。捕获20个精子细胞可以得到完整的STR分型,检出率为80%。随着精子数目增多,检出率提高而等位基因丢失率降低。30个精子检出率最高,为95%。结论激光捕获显微分离联用FISH技术可用于混合斑中男女细胞DNA的分离检验。

Objective To establish a method for isolating spermatozoa from epithelial cells in male-female mixtures by fluorescence in situ hybridization and laser capture microdissection. Methods Male sperm and female vaginal epithelial cells from two volunteers were collected and mixed with each other, CEPX SpectrumOrangeTM Y SpectrumGreenTMkits was used to hybridize the mixtures, and then the male and female cells were collected selectively by laser capture microdissection. Finally, a multiplex PCR was performed by the Identifiler kit using the on-chip LV PCR. The cells found to contain only a single orange（X）or a single green（Y） were classed as spermatozoa, where as cells found to contain both an orange and a green were considered as epithelial ceils. Results The completed STR typing was obtained from 20 sperm cells. The more ceils were analyzed, the higher recovery rates and lower allelic dropout rates were got. With 30 sperm cells, the highest recovery rate （95%） was obtained. Conclusions The combination of Fluorescence in situ hybridization and laser capture microdissection is effective for genotyping sperm from sperm/epithelial cell mixtures.