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目的建立用于显微捕获单细胞技术的龙胆紫染色法并评价其应用效果。方法制作20枚口腔拭子脱落细胞悬液,配制0.05g/mL龙胆紫染液。取100μL口腔细胞悬液加入0.15、0.25、0.5、1.0、2.0μL染色液,考察最佳染色浓度;分别染色5、10、30、60min,考察最佳染色时间;用最佳条件染色后抓捕3个细胞,采用联合LV-PCR技术扩增并进行DNA分型检测,进行重复试验20次,并对同一检材未经染色的抓捕细胞进行检测,用于比对STR分型成功率。将龙胆紫染色法用于案例检材的口腔上皮细胞分离检验。结果 100μL细胞悬液加入0.5μL 0.05g/mL龙胆紫染液,染色5min,胞核呈紫红色,与胞浆对比明显;染色时间延长不影响染色效果及细胞分离检验结果。优化后的龙胆紫染色法对低体积扩增无明显抑制(P>0.05)。应用此染色法于案例检材,胞核标识清晰,STR分型结果达同一认定标准。结论龙胆紫染色法利于细胞核的发现,有助于提高显微捕获单细胞技术的检测效能。
Objective To establish a gentian violet staining method for micro-capture single cell technology and evaluate its application effect. Methods 20 oral swabs were made into exfoliated cell suspensions to prepare 0.05g / mL gentian violet dye solution. 100μL oral cell suspension was added 0.15,0.25,0.5,1.0,2.0μL staining solution to investigate the best staining concentration; respectively staining 5,10,30,60 min, to investigate the best staining time; with the best conditions after staining 3 cells were amplified by a combination of LV-PCR technology and DNA typing test, the test was repeated 20 times, and the same sample of non-stained capture cells were detected for comparison of the success rate of STR typing. Gentian violet staining was used to test the oral epithelial cells isolated from samples. Results 100μL cell suspension was added with 0.5μL 0.05g / mL gentian violet stain. After staining for 5min, the nucleus was purple-red, showing obvious contrast with cytoplasm. The prolonged dyeing time did not affect the staining results and cell separation test results. The optimized gentian violet staining showed no significant inhibition on low volume amplification (P> 0.05). Application of this staining method in the case of samples, clear nuclear markers, STR typing results up to the same standard. Conclusion Gentian violet staining is beneficial to the discovery of nuclei and is helpful to improve the detection efficiency of single-cell micro-capture technique.