不同保存时间和不同精子数量精斑样本DNA分型比较

Comparative study of different sperm number and preserved time on genotyping

目的比较不同保存时间和不同精子数量精斑样本DNA分型的效果。方法制备精斑样本,保存10d的样本采用激光显微捕获30、20、15、10、5、1个精子,用于不同数量精子分型比较;保存10d、214d、375d的样本分别捕获30、20、10个精子,用于不同保存时间分型比较。比较各组检出率、等位基因丢失率和非特异性扩增率,采用χ2检验进行差异比较。结果①不同精子数量分型:捕获10个精子即可得到完整的DNA分型,且随着精子数增多,检出率逐渐提高而等位基因丢失率逐渐降低,30个精子等位基因丢失率为0%,1个精子则可达58.89%;②不同保存时间分型:总趋势是保存时间越短,捕获精子越多检出率越高,10个精子与20、30个精子组比较,均有显著性差异(P<0.05);等位基因丢失率及非特异性扩增率则随保存时间的延长而增加,相同保存时间的不同精子数量组之间和相同的精子数量的不同保存时间组之间比较,差异均具有统计学意义(P<0.05)。结论激光显微捕获精子数目和检材保存时间对DNA分型结果有直接影响。

Objective To compare the affection of different number and preserved time in studying DNA profile of sperm stains using singlecell separation technique.Methods Preparing sperm smear,6 groups of sperm（ 30,20,15,10,5,1sperm） were captured from the sample,which have been preserved for 10 days,by PLAM laser capture microdissection system so as to compare the affection of sperm number on genotyping.For the affection of preserved time,3 samples preserved for 10d,214d,375d were used,30,20,10sperm were separately captured from the identical sample.Then the DNA were amplified by IdentifilerPlus kit,finally the artificial alleles,detection rate and allelic dropout rate were compared with each group using the chi-square test.Results ①The results of different number of sperm： 10 sperm can be genotyped completely.As the number of cells increased,the detection rates were increased,whereas the allelic dropout rates were decreased.The detection rate of 30 sperm was 100%,allelic drop-out rate was 0%.For 1 sperm,the allelic drop-out rate can up to 58.89%; ②The results of different preserved time： there was a trend that the shorter preserved time and the more number of captured sperm,the detection rate was higher,currently,the rate between 10 sperm group and 20,30 sperm group both had significant differences（ P 0.05）; with the extension of preserved time,both the allelic drop-out rates and the non-specific amplification rates became higher.There were significant differences（ P 0.05） between the groups of the same preserved time but different sperm number,and the same sperm number but different preserved time.Conclusion The sperm number and preserved time,captured by laser microdissection,have a direct impact on genotyping.