Technique and its significance of detecting colorectal cancer cells by fluorescent amplification catalyzed with T7 RNA polymerase / 解放军医学杂志

ABSTRACT

Objective To develop a new method for detecting colorectal cancer cells in peripheral blood using a hybrid technology referred to as fluorescent amplification catalyzed with T7 polymerase technique (FACTT). Method A model of colorectal cancer circulating tumor cells in peripheral blood was reproduced by mixing serial dilutions of HT29 cells with 1?107 peripheral blood monocytes from healthy subjects. After all nucleated cells were isolated from the model, biotinylated monoclonal antibody CEA against the Gold epitopes Ⅳ, which was expressed on the cell surface of human colorectal cancer cells but not on the normal cells, was added followed by the adding of avidin-biotinylated-DNA. Then T7 RNA polymerase and nucleoside triphosphates (NTPs) were added to perform RNA amplification at room temperature for 3h. Finally, RNA products were quantified by adding the RNA intercalating dye RiboGreen. RT-PCR was used to detect the expression of CEA mRNA as a control to assess the sensitivity of fluorescent amplification catalyzed with T7 polymerase technique. Results A method of detecting circulating colorectal cancer cells in peripheral blood by fluorescent amplification catalyzed with T7 polymerase technique was established. It was shown that the technique had a detection level of 5 HT29 cells among 1?107 monocytes, and it was more sensitive than RT-PCR technique, by which the level of detection was 1?102 cells among 1? 107 monocytes. Conclusion Fluorsescent amplification catalyzed with T7 polymerase technique is a new and sensitive technology for detecting circulating colorectal cancer cells in peripheral blood.