3种碱性染液对显微捕获联合低体积扩增技术的影响

发布时间：2018-01-05 02:35

本文关键词：3种碱性染液对显微捕获联合低体积扩增技术的影响　出处：《重庆医科大学学报》2011年05期 　论文类型：期刊论文

【摘要】：目的:比较3种碱性染液对显微捕获联合低体积扩增(Lowvolume polymerase chain reaction,LVPCR)技术的影响,为细胞分离检验中细胞核标记物的选择提供依据。方法:显微捕获联合LVPCR中,用苏木素、龙胆紫、亚甲基蓝3种染液对口腔上皮细胞进行标识。参照龙胆紫染色法,比较0.5、1、2、5、8μl苏木素染液及2、5、8、10、15μl亚甲基蓝染液的染色效果,优化染色条件;并比较优化后的细胞核标识效果。显微操作仪捕获3种染液染色后的细胞进行DNA检验,比较检测结果;并捕获染色10、30、60 min口腔上皮细胞,比较染色时间对DNA检测结果的影响。结果:100μl口腔上皮细胞悬液加入2μl苏木素、0.5μl龙胆紫或5μl亚甲基蓝染液,细胞核均着色明显,与胞浆对比清晰。5个细胞的分离检验结果示有效分型率分别为90%、100%、66.7%,成功分型率为20%、100%、11.1%;龙胆紫、苏木素染色时间(≤60 min)对DNA检测结果无明显影响,而亚甲基蓝染色时间延长导致等位基因丢失等现象加重,影响检测结果。结论:3种染液均可清晰标识细胞核;而龙胆紫染液对LVPCR技术抑制最弱,为三者中较优的细胞核标记物。
[Abstract]:Objective: To compare the 3 kinds of alkaline solution of microdissection combined with low volume amplification (Lowvolume polymerase chain reaction, LVPCR) the impact of technology, provide the basis for cell separation and cell nucleus marker selection in the test. Methods: microdissection combined with hematoxylin eosin, LVPCR, gentian violet, methylene blue dye to identify 3 kinds of oral epithelial cells. According to crystal violet staining method, dyeing effect of the 0.5,1,2,5,8 L and 2,5,8,10,15 l hematoxylin dye methylene blue dye, optimal dyeing conditions; cell identification results and to compare the optimized micro operation instrument. Capturing 3 staining of cells by using DNA test, comparison of test results and capture 10,30,60 min staining; oral epithelial cells, staining time impact on the results of DNA. Results: 100 L oral epithelial cell suspension into 2 l hematoxylin, 0.5 L or 5 L methylene blue gentian violet liquid, The nuclei were stained in cytoplasm and obvious contrast result in clear separation and inspection.5 cells showed effective typing rate were 90%, 100%, 66.7%, classification success rate was 20%, 100%, 11.1%; longdanzi hematoxylin staining time (less than 60 min) had no obvious effect on the detection results of DNA and methylene blue prolonged increase allele loss, affect the detection results. Conclusion: the 3 kinds of dye can be clearly identified and gentian violet stain nuclei; weakest inhibition on LVPCR technology, nuclear marker is the best in the three.

【作者单位】：重庆医科大学基础医学院法医学教研室;公安部物证鉴定中心法医遗传学重点实验室;中国人民公安大学刑事科学技术系;重庆市公安局技术处法医科;
【基金】：“十一·五”国家科技支撑计划资助项目(编号:2006BAK07B01)
【分类号】：D919.2
【正文快照】： 细胞捕获和低体积扩增(Low volume polymerase chainreaction,LVPCR)技术[1~4]是细胞分离检验的两大核心。尤其是LVPCR,其高灵敏度降低了分离检验中细胞数量的要求,但反应体积的减少,亦降低了抗干扰的能力。随着细胞分离检验技术在实际检案中的推广应用,细胞标识方法的引入成

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