Breast cancer 21 gene detection kit

A gene detection and kit technology, applied in the field of breast cancer 21 gene detection kits, can solve the problems of long time required for obtaining results, low false positives, long time consumption, etc. The effect of saving time and cost

Inactive Publication Date: 2020-08-07
深圳市草履虫生物科技有限公司
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  • Abstract
  • Description
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Problems solved by technology

[0003] At present, there are two main types of kits for breast cancer detection on the market. One is the traditional fluorescent quantitative PCR to detect the expression level of 21 genes. Although the effect of this method is well evaluated, it takes a long time and takes a long time to obtain the results. ; The other is in situ hybridization detection, which also has good effects and low false positives, but it also takes a long time

Method used

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Examples

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Effect test

Embodiment 121

[0086] Example 1 Design of PCR primers and detection probes for 21 genes

[0087] Find the protein sequences of 21 genes on uniprot, find the corresponding information on NCBI from Refseq, find the gene nucleotide sequence from CDS, and design primers and probes according to the primer design and probe design principles of constant temperature fluorescent amplification PCR. Probes, where the principle of probe design includes that the first sequence at the 5' end cannot be a G base, and the last sequence at the 3' end must be an A base, avoiding the first four sequences at the 3' end containing 3 or more G bases base, to avoid the middle region of the probe containing two or more consecutive C bases, the designed primers and probe sequences are shown in Table 1 below, where FAM, HEX, ROX are fluorescent groups, THF is tetrahydrofuran, BHQ1, BHQ2 For the quenching group, the number of bases between the fluorescent group, THF and the quenching group can be 0, 1 or 2.

[0088] T...

Embodiment 2

[0092] Example 2 Constant temperature fluorescent quantitative PCR detection sample 21 gene levels

[0093] Process paraffin-embedded breast cancer specimens, extract total mRNA from them, take 2 μl for gel electrophoresis, and the results are as follows: figure 1 As shown, lanes 1 and 2 are the results of sample mRNA. The mRNA was then reversed into cDNA using the Quantum Gold One-Step Reverse Transcription Kit. Using cDNA as a template to detect the expression level of 21 genes in breast cancer samples, through the optimized combination of specific primers and fluorescent probes in the present invention, the accurate, simple and rapid simultaneous detection of 21 gene expression levels can be realized. At the same time, using DNA plasmids of 21 genes as positive control templates, a reaction system for detecting the expression levels of 21 genes by constant temperature multiplex fluorescent PCR was established, and finally the Ct value was used to calculate the RS value as ...

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Abstract

The invention provides a breast cancer 21 gene detection kit. A constant-temperature fluorescent quantitative PCR amplification enzyme mixed solution is added into a breast cancer 21 gene detection kit, the newest constant-temperature fluorescent quantitative PCR amplification technology is introduced, the problem that traditional fluorescent quantitative PCR consumes long time is solved, the whole amplification time is shortened to 40 min to 1 h from 2 h to 3 h, operation is simpler and more convenient than that of a traditional method, and the time cost of medical inspectors is greatly saved. Meanwhile, the time for doctors and patients to wait for results is shortened, and an optimal scheme can be provided for subsequent chemotherapy of the patients earlier and faster.

Description

technical field [0001] The invention relates to the field of in vitro diagnosis, in particular to a breast cancer 21 gene detection kit. Background technique [0002] A 21-gene expression assay, including 16 cancer-related genes and 5 reference genes, was developed as a multigene array to assess residual risk after surgery in patients with HR-positive, HER2-negative, and lymph node-negative early breast cancer. The 16 cancer-related genes are divided into five groups: 1. Cell proliferation-related genes: Ki67, STK15, Survivin, CCNB1 (cyclin B1), MYBL2; 2. Cell invasion-related genes: MMP11 (stromolysin 3), CTSL2 (cathepsinL2); 3 1. Estrogen-related genes: ER, PGR, BCL2, SCUBE2; 4. HER2-related genes: GRB7 and HER2 (ERBB2); 5. Three independent genes: GSTM1, CD68, BAG1; 6. 5 internal reference genes: ACTB ( b-actin), GAPDH, RPLPO, GUS, TFRC. This test can provide prediction of treatment effect and 10-year recurrence risk prediction for the prognosis of early breast cancer p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6844
CPCC12Q1/6844C12Q2531/119C12Q2563/107C12Q2522/101
Inventor 王坤谭毅彬钱凯林冰
Owner 深圳市草履虫生物科技有限公司
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