压力循环技术用于人指甲DNA提取及方法学评价

Evaluation of the methodology for applying Pressure Cycling Technology in nuclear DNA genotyping extraction from human nail

目的将压力循环技术（PCT）用于指甲DNA提取,并对方法学进行评价。方法收集10份人指甲样本,剪碎约为1mm×1mm大小,采用10%漂白粉水,10%SDS,10%漂白粉水,无菌水清洗样本。10份样本各分成两组,1组用压力循环技术处理,另1组不作处理,提取DNA经复合扩增并进行STR分型检测,用于评价压力循环技术的作用。取5份指甲样本用血浸泡,5份用去离子水浸泡,之后采用上述清洗方法各清洗1-3次,收集各次清洗用的无菌水提取DNA,经STR分型检测,用于评价清洗对去除外源性DNA的效果。结果 10份经压力循环技术处理的样本中有7例比相应未经处理样本DNA提取量更高,但两组进行统计学处理,差异不具有统计学意义（P〉0.05）;两组样本中提取DNA含量在0.026 ng以上的样本均得到完整的STR分型,与相应口腔拭子样本对照准确无误。血污染和非血污染样本清洗二次以上,均可避免外源性DNA的污染。结论使用压力循环技术并配合本文清洗方法,可有效提高人指甲DNA的提取效率,并避免外源性生物DNA的干扰,保证DNA分型结果的准确。

Objective To apply pressure cycling technology（ PCT） in nuclear DNA extraction from human nails, and complete the evaluation of corresponding extraction methodology. Methods Nails samples collected from 10 volunteers were divided into two groups（ 10 samples for each group）. Both groups of samples were cut into 1mm by 1mm pieces,and then cleaned with sterile water which was prepared in 10 % bleaching powder,10 % SDS. The group of samples served as experimental group was processed with pressure cycling technology,while control group was not. DNA extracted from both experimental group and control group were used for multiplex amplification and STR typing,to evaluate the effect of pressure cycling technology. Two additional groups containing 5 human nail samples each were set to evaluate the impact of aforementioned cleaning methods on exogenous DNA removal. The experiment group was immersed in blood,while the control group was immersed in deionized water. Results 7 out of 10 samples in experimental group shown higher DNA extraction yield than corresponding control group, but the statistical treatment indicates there is no statistically significant difference between experimental group and control group（ P 0. 05）; Samples with DNA extraction 0. 026 ng in both groups got complete STR typing,and the results were exactly aligned with corresponding mouth swab samples. Samples with or without blood contamination can avoid exogenous DNA contamination by applying aforementioned cleaning methods more than twice. Conclusion PCT along with aforementioned cleaning methods can significantly improve the efficiency of DNA extraction from human nails,and the accuracy of DNA genotyping can be ensured by litter impact from exogenous DNA contamination.