Primer set for intraoperatively and assistedly identifying whether sentinel node generates breast cancer cell metastasis, and application thereof
A breast cancer cell and sentinel lymph node technology, which is applied in biochemical equipment and methods, recombinant DNA technology, and microbial determination/inspection, etc. problems, to achieve the effect of good clinical application value, avoid pollution, and short response time
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Embodiment 1
[0049] Embodiment 1, design and screening of primer set
[0050] Design and synthesize primer compositions for identifying the CK19 gene (6 groups in total, all of which are LAMP primer compositions, consisting of FIP, BIP, F3 and R3), and primer compositions for identifying the PIP gene (4 groups in total, Both are LAMP primer compositions, all composed of FIP, BIP, F3 and R3) and primer compositions for identifying PBGD genes (3 groups in total, all are LAMP primer compositions, all composed of FIP, BIP, F3 and R3 ).
[0051] 1. Use MCF-7 cells and NAMALWA cells for primer screening:
[0052] 1. Use Tiangen Biochemical Technology (Beijing) Co., Ltd. Cell / Bacteria Total RNA Extraction Kit (Product No.: DP430) to extract total RNA from MCF-7 cells and NAMALWA cells, and freeze them at -80°C.
[0053] 2. Using the total RNA solution extracted in step 1 as a template, carry out RT-LAMP with each set of primer compositions.
[0054] RT-LAMP reaction system (25 μL): 12.5 μl of ...
Embodiment 2
[0118] Embodiment 2, detect the sensitivity of the target gene in the cell by Loopamp turbidimeter
[0119] 1. Use Tiangen Biochemical Technology (Beijing) Co., Ltd. Cell / Bacterial Total RNA Extraction Kit (Product No.: DP430) to extract the total RNA of MCF-7 cells, and freeze at -80°C.
[0120] 2. The total RNA solution obtained in step 1 was serially diluted with deionized water to obtain RNA concentrations of 1 ng / μl, 10 -1 ng / μl, 10 -2 ng / μl, 10 -3 ng / μl, 10 -4 ng / μl, 10 -5 ng / μl, 10 -6 ng / μl and 10 -7 ng / μl dilution.
[0121] 3. Using the total RNA solution extracted in step 1 as a template, perform RT-LAMP of the three systems respectively.
[0122] The difference between the RT-LAMP reaction system and the RT-LAMP reaction system in Step 3 of Example 1 is that 5 μl of the dilution prepared in Step 2 was used instead of 5 μl of the total RNA solution.
[0123] The RT-LAMP reaction procedure is the same as Step 3 of Example 1.
[0124] Real-time detection of the...
Embodiment 3
[0128] Embodiment 3, detect clinical sample by Loopamp turbidimeter and compare with GeneSearch method
[0129] 1. Use the kit (QIAGEN, RNeasy Mini Kit (250), item number: 74106) to extract the total RNA of the sentinel lymph node of the hospital-diagnosed breast cancer patients, and perform the detection by GeneSearch method (Blumencranz P, Whitworth PW, et al. Scientific Impact Recognition Award.Sentinel node staging for breast cancer:intraoperative molecular pathology overcomes conventional histologic sampling errors.Am J Surg.2007Oct;194(4):426-32.), the remaining total RNA was frozen at -80℃.
[0130] 2. Using the total RNA solution extracted in step 1 as a template, perform RT-LAMP of the three systems respectively.
[0131] The RT-LAMP reaction system and RT-LAMP reaction procedure are the same as Step 3 of Example 1.
[0132] According to the comparative analysis with the results of the first 50 cases of GeneSearch, confirm the threshold value of RT-LAMP detection (th...
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