Abstract:

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.

Key words: loop-mediated isothermal amplification, cytokeratin 19, breast neoplasms, sentinel lymph node, metastasis