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

Inactive Publication Date: 2014-01-01
AFFILIATED HOSPITAL CHINA ACADEMY OF MILITARY MEDICAL SCI +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The disadvantage of this method is that the false negative rate is high (5-52%), and the section is easily distorted and deformed due to the influence of surrounding fat tissue, making it difficult to observe
Although paraffin section is currently the gold standard for judging whether SLN has metastasized, it takes 1-2 days to wait for the result, which is not conducive to intraoperative
If this method is adopted, the operation of the patient needs to be performed twice: the first operation is to remove the SLN, and the second operation can only be decided after the results of the paraffin section come out, which will cause a lot of unnecessary troubles for the patient and the hospital
[0007] In addition, the above two slicing methods still have some common shortcomings: ① In order to save manpower and material resources, generally only 2-5% of the lymph nodes are cut for testing, and different slicing positions may lead to different results; ② During the slicing process, There is a thickness interval, and metastases below this interval are likely to be missed; ③The slice method provides subjective results, and different pathologists may get different results for the same slice
[0009] Obviously, conventional pathological methods cannot fully meet the above requirements

Method used

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  • Primer set for intraoperatively and assistedly identifying whether sentinel node generates breast cancer cell metastasis, and application thereof
  • Primer set for intraoperatively and assistedly identifying whether sentinel node generates breast cancer cell metastasis, and application thereof
  • Primer set for intraoperatively and assistedly identifying whether sentinel node generates breast cancer cell metastasis, and application thereof

Examples

Experimental program
Comparison scheme
Effect test

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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Abstract

The present invention discloses a primer set for intraoperatively and assistedly identifying whether sentinel node generates breast cancer cell metastasis, and an application thereof. The present invention provides a primer set, which comprises a primer composition 1, a primer composition 2 and a primer composition 3, wherein the primer composition 1 comprises single-stranded DNA molecules represents by sequences 1-6, the primer composition 2 comprises single-stranded DNA molecules represents by sequences 7-12, and the primer composition 3 comprises single-stranded DNA molecules represents by sequences 13-18. With application of the primer set to identify whether sentinel node generates breast cancer cell metastasis, SLN metastasis status can be judged during a surgery process, such that secondary surgery of patients can be avoided so as to reduce suffering of patients, substantially save hospital expenses, and provide important clinical and social significance.

Description

technical field [0001] The invention relates to a primer set and application thereof for assisting in identifying whether breast cancer cell metastasis occurs in sentinel lymph nodes during operation. Background technique [0002] Breast cancer is a common female tumor, and its morbidity and mortality rank first and second among female tumors. Surgery is one of the most commonly used treatment methods for breast cancer patients. In the more than 100 years since Halsted founded the radical mastectomy in 1894, the surgical treatment of breast cancer has undergone attempts and revisions of extended radical mastectomy and modified radical mastectomy. The biggest change is undoubtedly the rapid development of breast-conserving therapy in the last 30 years of the 20th century. This makes the surgical treatment mode change from "maximum tolerable treatment" to "minimum effective treatment", and begins to emphasize conservative treatment focusing on quality of life. [0003] The gr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6844C12Q2531/119
Inventor 刘毅郝晓鹏尉承泽
Owner AFFILIATED HOSPITAL CHINA ACADEMY OF MILITARY MEDICAL SCI
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