目的

建立快速准确、全面客观的判断前哨淋巴结转移状态的分子病理检查方法。

方法

合成细胞角蛋白19(CK19)的环介导等温扩增(LAMP)引物,应用LAMP 技术扩增乳腺癌细胞MCF-7 的cDNA,验证引物序列的可靠性;通过RT-LAMP 技术分别检测MCF-7、Daudi、Namawal 细胞的RNA,验证实验体系的可行性;将手术获取的乳腺癌患者前哨淋巴结行RT-LAMP 检测CK19 表达,与常规病理学方法的检测结果相对比,最终通过荧光定量以及离心观测沉淀等手段缩短整体检测时间。

结果

应用LAMP 技术能够检测到MCF-7 乳腺癌细胞的CK19 表达,而Daudi、Namawal 细胞CK19的表达均为阴性;经病理检查证实为癌转移阳性的3 枚前哨淋巴结,通过RT-LAMP 均检测到CK19 表达;经荧光定量及离心观测沉淀等手段优化后反应时间可以缩短至1 h之内。

结论

LAMP 检测CK19 表达,能够准确区分上皮来源细胞和淋巴瘤细胞,检测转移阳性淋巴结的结果与常规病理学结果完全符合,其具有简便、准确、快速等优点。

Objective

To establish a rapid and accurate method to objectively detect the metastasis of sentinel lymph node in breast cancer.

Methods

The loop-mediated isothermal amplification (LAMP) primers of cytokeratin 19 (CK19) was synthetized, LAMP was applied to amplify breast cancer cell line MCF-7 cDNA, and the reliability of the primer sequences was validated. RT-LAMP was applied to detect the RNA sequence in MCF-7,Daudi, and Namawal cells for the further verification of experiment system. Sentinel lymph nodes from breast cancer patients, which were dissected in surgery, underwent RT-LAMP for detection of CK19 expression, then the results were compared with those in routine pathological tests. The overall detection time can be shortened by fluorescence quantitative method and observing precipitation after centrifugation.

Results

The CK19 expression in breast cancer MCF-7 cells was detected by LAMP while CK19 expression were negative in Daudi, and Namawal cells. Three positive sentinel lymph node with cancer metastasis confirmed by pathological examination presented CK19 expression by RT-LAMP. The reaction time was shortened to within 1 h by the optimization of fluorescent quantitative method and observing precipitation after centrifugation.

Conclusions

LAMP for detection of CK19 expression can accurately distinguish lymphoma cells from epithelial cells, and the detection results are fully in line with the routine pathological results. This method is simple,accurate and fast.