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A digital PCR method and detection kit for detecting HER2 copy number variation in breast cancer

A detection kit, copy number variation technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve problems such as the inability to completely and accurately determine HER2, and achieve high sensitivity and repeatability, high sensitivity, high The effect of accuracy

Active Publication Date: 2022-08-05
NAT INST OF METROLOGY CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the technical solution of this patent application can only solve the problem of HER2-positive samples that are judged as suspected positive by conventional methods, but cannot solve the samples that are actually false negatives even if judged to be negative by conventional methods. Such samples are due to chromosome doubling , so neither the conventional method nor the method of this patent application can completely and accurately judge HER2 positive

Method used

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  • A digital PCR method and detection kit for detecting HER2 copy number variation in breast cancer
  • A digital PCR method and detection kit for detecting HER2 copy number variation in breast cancer
  • A digital PCR method and detection kit for detecting HER2 copy number variation in breast cancer

Examples

Experimental program
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Effect test

Embodiment 1

[0088] Example 1 Detection of normal human cell DNA and breast cancer cell DNA by ddPCR technology

[0089] (1) Extracting cellular genomic DNA:

[0090] Centrifuge the sample, add buffer to suspend, and then add proteinase K to dissolve the sample to obtain a sample lysate; add 200 μL of buffer, invert and mix well, and place at 70°C for 10 minutes; add 200 μL of absolute ethanol, fully Shake and mix for 15 seconds; add the obtained solution and the flocculent precipitate to an adsorption column, centrifuge at 12,000 rpm (~13,400 × g) for 30 seconds, pour off the waste liquid, and put the adsorption column back into the collection tube; add to the adsorption column 500μL buffer, centrifuge at 12000rpm (~13400×g) for 30 seconds, pour off the waste liquid, put the adsorption column into the collection tube; add 600μL of rinsing solution to the adsorption column, centrifuge at 12000rpm (~13400×g) for 30 seconds, pour Discard the waste liquid, put the adsorption column into the ...

Embodiment 2

[0103] Example 2 Using ddPCR technology to detect samples with different mutation levels of HER2

[0104] Samples 3 and 6 were mixed in different proportions to configure 4 samples with different mutation levels of HER2, and the H1 / P1, H2 / P1, and H3 / P1 values ​​of each sample were determined by the method of Example 1. The results are shown in Table 2 below. The ratios of copy number and internal reference gene amplified by the three pairs of primers were very close to the theoretical value prepared, the correlation coefficients were all greater than 0.93, and the linear correlation coefficients were all greater than 0.995. It shows that the method described in the present invention has high accuracy, and the detection results of the lowest mutation level sample 3 show that the method can accurately detect samples with a copy increase as low as 1, and the determination results of the three pairs of primers are highly consistent.

[0105] Table 2 Determination results of sampl...

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Abstract

The invention discloses a digital PCR method and a detection kit for detecting HER2 copy number variation in breast cancer. The kit includes at least one pair of primers for detecting HER2 gene copy number variation and a matched probe. , and a pair of primers and a matching probe for the internal reference gene used for detection. The method of the present invention adopts three sets of specific primer probes for HER2 gene and one set of internal reference genes, thereby effectively eliminating false negative results caused by amplification of a single primer probe, so compared with the prior art, it has Higher accuracy and lower uncertainty interval of 1.8‑2.2 for existing methods to 1.0‑1.2, with higher sensitivity.

Description

technical field [0001] The invention relates to the technical field of gene detection related to tumor targeted therapy, in particular to a digital PCR method and a detection kit for detecting HER2 copy number variation in breast cancer. Background technique [0002] Breast cancer is one of the most common malignant tumors in women, with more than 1 million new cases every year. It is a disease that seriously threatens the health and even life of patients. The treatment effect and prognosis of different patients may also vary greatly. [0003] HER2 gene is a proto-oncogene located on the long arm of human chromosome 17, encoding human epidermal growth factor receptor-2 (human epidermal growth factor receptor-2, HER2) with receptor tyrosine kinase (PTK) activity. Membrane glycoprotein, a member of the epidermal growth factor receptor family, needs to combine with other receptors in the family to form a heterodimer to play a signal transduction function. Overexpression or am...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6858
CPCC12Q1/6858C12Q2563/107C12Q2563/159C12Q2545/101C12Q2537/16
Inventor 董莲华王霞王晶
Owner NAT INST OF METROLOGY CHINA