单细胞分离荧光原位杂交法用于男女混合血DNA分型

DNA profile of male and female cells in blood mixture by single cell separation and fluorescence in situ hybridization technique

目的采用单细胞分离荧光原位杂交法精确分离混合血样中男性和女性细胞并进行分型检验。方法收集男、女性血,按照男∶女为1∶5、1∶10、1∶20制备混合血样,加入0.075mol/L KCl 600μL,轻混、放置30min后加入150μL固定液离心留沉淀涂片,利用Vysis 30-161050试剂盒进行荧光原位杂交,并用PALM激光显微捕获系统分离出男、女性细胞,使用Identifiler试剂盒复合扩增并进行检测。结果捕获8个血细胞即可得到完整的DNA分型,且随着细胞数目的增多,检出率逐渐提高而等位基因丢失率逐渐降低。10个血细胞的检出率最高,为93.75%。5μL男性血液与本实验各比例女性血混合用本文方法检验均可获得男性分型。案例混合血斑经检验获得单一男性和女性分型。结论单细胞分离荧光原位杂交法可用于男女混合血样本中DNA分型检验。

Objective Using single cell separation and fluorescence in situ hybridization technique to accurately separate male and female cells and to test DNA profile in blood mixture.Methods male and female blood were collected,then mixed blood samples were prepared as male and female in accordance with 1∶ 5,1∶ 10,1∶ 20.The samples were sequentially added 600μL 0.75mol/L KCl,agitated for 30min,followed by 150μL fixative,then centrifuged before being applied to membrane slides.The slides were hybridized by Vysis 30-161050 kit.Male and female cells were isolated by PLAM laser capture microdissection system.Finally,a multiplex polymerase chain reaction was performed using the Identifiler kit.Results 8 blood cells can be genotyped completely.As the number of cells analyzed was increased,the call rates were increased,whereas the allelic dropout rates were decreased.The detection rate of 10 blood cells,which was the highest rate,was 93.75%.Genetic profile of male can be obtained from 5μL male blood and female blood mixed in different rates by above method.Single genetic profiles of male and female also can be obtained in a blood stain casework sample.Conclusion Single cell separation and fluorescence in situ hybridization technique can be used to get profiles of male and female contribution in blood mixture.