Method and detection kit for detecting ERBB2 gene amplification based on digital PCR technology

Abstract

The invention belongs to the technical field of molecular diagnosis, and particularly relates to a method and detection kit for detecting ERBB2 gene amplification based on a digital PCR technology. The kit comprises primer and probe premix liquid, wherein the premix liquid comprises upstream and downstream primers for detecting an ERBB2 gene, a fluorescent probe for detecting the ERBB2 gene, upstream and downstream primers for detecting a human ATCB reference gene, and a fluorescent probe for detecting the human ATCB reference gene. The method comprises the following steps of S1, providing a detection sample of a testee, wherein the detection sample is a plasma sample; S2, extracting ctDNA of a to-be-detected sample from the plasma sample in the step S1; S3, performing an amplification reaction on the sample extracted in the step S2 by using a digital PCR platform; and S4, analyzing and interpreting an amplification result. The ERBB2 gene amplification detection method provided by theinvention is based on a micro-droplet digital PCR detection system, reduces the interference of manual operation, has the advantages of stable result, high accuracy and good data amplification effect,and can be more conveniently used for auxiliary diagnosis and clinical treatment guidance.

Description

technical field

[0001] The invention belongs to the technical field of molecular diagnosis, in particular to a method for detecting ERBB2 gene amplification based on digital PCR technology and a detection kit thereof. Background technique

[0002] The tyrosine kinase receptor ERBB2 (also known as ERBB2 / NEU) gene encodes a 185kDa transmembrane glycoprotein with tyrosine kinase activity and is a member of the EGFR family. Amplification or overexpression of the ERBB2 gene can lead to excessive proliferation of cells and the formation of tumors. Studies have shown that about 25% to 30% of invasive breast cancer and 80% of gastric cancer patients have ERBB2 gene amplification or protein overexpression.

[0003] ERBB2 amplification may be used to predict the body's sensitivity to ERBB2-targeted therapeutic drugs, such drugs include lapatinib, trastuzumab, pertuzumab, T-DM1, etc. Taking the earliest and most successful trastuzumab (Herceptin) as an example, clinical studies have ...

Examples

Embodiment 1

[0045] Detection of ERBB2 gene amplification in plasma samples from patients with gastric cancer by ddPCR.

[0046] 1. The steps of micro-droplet digital PCR detection are as follows:

[0047] 1) Preparation of samples: The source of sample DNA is the blood samples of patients with gastric cancer. The DNA sample can be properly diluted to ensure that the sample signal copy number is between 1×102 copy / μL and 1×105 copy / μL.

[0048]2) Prepare the PCR reaction solution according to the following ratio: 2×ddPCR Master Mix, primers, probes, 1 μL cfDNA template, make up to a final volume of 20 μL with distilled water, and configure a digital PCR mixture, in which the content of each primer is 400 nM , each probe is 500nM, and the cfDNA template is 1-10ng / μL.

[0049] 3) Load the prepared 20 μL digital PCR reaction solution onto the chip.

[0050] 4) Put the chip into the PCR machine, and carry out the amplification reaction according to the following conditions: pre-denaturation...

Embodiment 2

[0057] 1. Based on ddPCR technology, compare the consistency of ERBB2 gene amplification between plasma ctDNA and tumor tissue DNA in patients with gastric cancer.

technical field

[0001] The invention belongs to the technical field of molecular diagnosis, in particular to a method for detecting ERBB2 gene amplification based on digital PCR technology and a detection kit thereof. Background technique

[0002] The tyrosine kinase receptor ERBB2 (also known as ERBB2 / NEU) gene encodes a 185kDa transmembrane glycoprotein with tyrosine kinase activity and is a member of the EGFR family. Amplification or overexpression of the ERBB2 gene can lead to excessive proliferation of cells and the formation of tumors. Studies have shown that about 25% to 30% of invasive breast cancer and 80% of gastric cancer patients have ERBB2 gene amplification or protein overexpression.

[0003] ERBB2 amplification may be used to predict the body's sensitivity to ERBB2-targeted therapeutic drugs, such drugs include lapatinib, trastuzumab, pertuzumab, T-DM1, etc. Taking the earliest and most successful trastuzumab (Herceptin) as an example, clinical studies have ...

Examples

Embodiment 1

[0045] Detection of ERBB2 gene amplification in plasma samples from patients with gastric cancer by ddPCR.

[0046] 1. The steps of micro-droplet digital PCR detection are as follows:

[0047] 1) Preparation of samples: The source of sample DNA is the blood samples of patients with gastric cancer. The DNA sample can be properly diluted to ensure that the sample signal copy number is between 1×102 copy / μL and 1×105 copy / μL.

[0048]2) Prepare the PCR reaction solution according to the following ratio: 2×ddPCR Master Mix, primers, probes, 1 μL cfDNA template, make up to a final volume of 20 μL with distilled water, and configure a digital PCR mixture, in which the content of each primer is 400 nM , each probe is 500nM, and the cfDNA template is 1-10ng / μL.

[0049] 3) Load the prepared 20 μL digital PCR reaction solution onto the chip.

[0050] 4) Put the chip into the PCR machine, and carry out the amplification reaction according to the following conditions: pre-denaturation...

Embodiment 2

[0057] 1. Based on ddPCR technology, compare the consistency of ERBB2 gene amplification between plasma ctDNA and tumor tissue DNA in patients with gastric cancer.

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