龙胆紫染色法在显微捕获单细胞分离技术中的应用

Application of gentian violet stain-method in single cell micromanipulation technique

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 evaluate its validity. Methods 20 establish gentian violet stain-method in single cell micromanipulation and oral swabs and 0.05g/mL gentian violet solution were prepared, and then 0.15 ,0. 25 ,0. 5 ,1 and 2μL 0.05g/mL gentian violet solution were added into 100mL oral epithelium cell suspension respectively to determine the optimal staining concentration. At the same time, the epithelium cells were stained for 5,10,30 and 60mins respectively to determine the optimal staining time. After stained with the optimal condition, three colored cells were captured and then tested using low volume PCR （LV-PCR） technique with 20 replications. The success rate of STR genotyping was compared between the stained samples and the same samples without staining. In addition, the gentian violet stain-method had a/so been applied to the oral epithelium cell micromanipulation in a criminal case. Results 0.5μL 0.05g/mL gentian violet solution added into 100μL cell suspension for 5rains could distinguish the nuclei from cytoplasm by staining them into violet, and staining time extension had no effect on the test results. No significant effect on the LV-PCR was found by the optimal gentian violet stain-method （P 〉 0.05）. Nuclei could he clearly marked in case samoleusing this stain-method and STR genotyping result could meet the standard of identity identification. Conclusion It is conducive to the discovery of the nucleus by gentian violet stain-method and thus can help improve the performance of single cell micromanipulation.